1d05 pcsk9 antagonists

ABSTRACT

Antagonists of human proprotein convertase subtilisin-kexin type 9 (“PCSK9”) are disclosed. The disclosed antagonists are effective in the inhibition of PCSK9 function and, accordingly, present desirable antagonists for use in the treatment of conditions associated with PCSK9 activity. The present invention also discloses nucleic acid encoding said antagonists, vectors, host cells, and compositions comprising the antagonists. Methods of making PCSK9-specific antagonists as well as methods of using the antagonists for inhibiting or antagonizing PCSK9 function are also disclosed and form important additional aspects of the present disclosure.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/322,867; filed Feb. 6, 2009; which is herein incorporated byreference in its entirety; and which claims the benefit of U.S.Provisional Application No. 61/063,949, filed on Feb. 7, 2008, and61/066,577, filed Feb. 21, 2008.

STATEMENT REGARDING FEDERALLY-SPONSORED R&D

Not Applicable.

REFERENCE TO MICROFICHE APPENDIX

Not Applicable.

BACKGROUND OF THE INVENTION

Proprotein convertase subtilisin-kexin type 9 (hereinafter called“PCSK9”), also known as neural apoptosis-regulated convertase 1(“NARC-1”), is a proteinase K-like subtilase identified as the 9^(th)member of the secretory subtilase family; see Seidah et al., 2003 PNAS100:928-933. The gene for PCSK9 localizes to human chromosome 1p33-p343;Seidah et al., supra. PCSK9 is expressed in cells capable ofproliferation and differentiation including, for example, hepatocytes,kidney mesenchymal cells, intestinal ileum, and colon epithelia as wellas embryonic brain telencephalon neurons; Seidah et al., supra.

Original synthesis of PCSK9 is in the form of an inactive enzymeprecursor, or zymogen, of ˜72-kDa which undergoes autocatalytic,intramolecular processing in the endoplasmic reticulum (“ER”) toactivate its functionality. This internal processing event has beenreported to occur at the SSVFAQ↓SIPWNL¹⁵⁸ motif (SEQ ID NOs: 19 and 20,respectively); Benjannet et al., 2004 J. Biol. Chem. 279:48865-48875.Such internal processing has been reported as a requirement of exit fromthe ER; Benjannet et al., supra; Seidah et al., supra. The cleaved and,thereby, activated protein is secreted in association with the cleavedpeptide; supra.

The sequence for human PCSK9 (˜22-kb long with 12 exons encoding a 692amino acid protein) can be found in one instance at Deposit No.NP_(—)777596.2. Human, mouse and rat PCSK9 nucleic acid sequences havebeen deposited; see, e.g., GenBank Accession Nos.: AX127530 (alsoAX207686), NP_(—)705793 (also Q80W65), and P59996, respectively. PCSK9possesses several domains found in other proprotein convertases,including an N-terminal signal sequence, a pro domain, a catalyticdomain and a cysteine-rich C terminal domain. The PCSK9 catalytic domainshares high sequence similarity with the proteinase K family ofsubtilases and, notably, a catalytic triad of D186, H226 and S386.

PCSK9 is disclosed and/or claimed in several patent publicationsincluding, but not limited to the following: PCT Publication Nos. WO01/31007, WO 01/57081, WO 02/14358, WO 01/98468, WO 02/102993, WO02/102994, WO 02/46383, WO 02/90526, WO 01/77137, and WO 01/34768; USPublication Nos. US 2004/0009553 and US 2003/0119038, and EuropeanPublication Nos. EP 1 440 981, EP 1 067 182, and EP 1 471 152.

PCSK9 has been ascribed a role in the differentiation of hepatic andneuronal cells (Seidah et al., supra.), is highly expressed in embryonicliver, and has been strongly implicated in cholesterol homeostasis.Studies have suggested a specific role for PCSK9 in cholesterolbiosynthesis or uptake. In a study of cholesterol-fed rats, Maxwell etal. found that PCSK9 was downregulated in a similar manner to threeother genes involved in cholesterol biosynthesis, Maxwell et al., 2003J. Lipid Res. 44:2109-2119. The expression of PCSK9 has, in fact, beenshown to be regulated by sterol regulatory element-binding proteins(“SREBP”), as seen with other genes involved in cholesterol metabolism;supra. Later support for these findings came about through a study ofPCSK9 transcriptional regulation which demonstrated that such regulationwas quite typical of other genes implicated in lipoprotein metabolism;Dubuc et al., 2004 Arterioscler. Thromb. Vasc. Biol. 24:1454-1459.Statins have been shown to upregulate PCSK9 expression in a mannerattributed to the cholesterol-lowering effects of the drugs; supra.Moreover, it has been shown that PCSK9 promoters possess two conservedsites involved in cholesterol regulation, a sterol regulatory elementand an Sp1 site; supra.

Several lines of evidence demonstrate that PCSK9, in particular, lowersthe amount of hepatic LDLR protein and thus compromises the liver'sability to remove LDL cholesterol from the circulation.Adenovirus-mediated overexpression of PCSK9 in the livers of miceresults in the accumulation of circulating LDL-C due to a dramatic lossof hepatic LDLR protein, with no effect on LDLR mRNA levels; Benjannetet al., 20041 Biol. Chem. 279:48865-48875; Maxwell & Breslow, 2004 PNAS101:7100-7105; Park et al., 2004 J. Biol. Chem. 279:50630-50638; andLalanne et al., 2005 J. Lipid Res. 46:1312-1319. The effect of PCSK9overexpression on raising circulating LDL-C levels in mice is completelydependent on the expression of LDLR, again, indicating that theregulation of LDL-C by PCSK9 is mediated through downregulation of LDLRprotein. In agreement with these findings, mice lacking PCSK9 or inwhich PCSK9 mRNA has been lowered by antisense oligonucleotideinhibitors have higher levels of hepatic LDLR protein and a greaterability to clear circulating LDL-C; Rashid et al., 2005 PNAS102:5374-5379; and Graham et al., 2007 J. Lipid Res. 48(4):763-767. Inaddition, lowering PCSK9 levels in cultured human hepatocytes by siRNAalso results in higher LDLR protein levels and an increased ability totake up LDL-C; Benjannet et al., 2004 J. Biol. Chem. 279:48865-48875;and Lalanne et al., 2005 J. Lipid Res. 46:1312-1319. Together, thesedata indicate that PCSK9 action leads to increased LDL-C by loweringLDLR protein levels.

A number of mutations in the gene PCSK9 have also been conclusivelyassociated with autosomal dominant hypercholesterolemia (“ADH”), aninherited metabolism disorder characterized by marked elevations of lowdensity lipoprotein (“LDL”) particles in the plasma which can lead topremature cardiovascular failure; see Abifadel et al., 2003 NatureGenetics 34:154-156; Timms et al, 2004 Hum. Genet. 114:349-353; Leren,2004 Clin. Genet. 65:419-422. A later-published study on the S127Rmutation of Abifadel et al., supra, reported that patients carrying sucha mutation exhibited higher total cholesterol and apoB100 in the plasmaattributed to (1) an overproduction of apoB100-containing lipoproteins,such as low density lipoprotein (“LDL”), very low density lipoprotein(“VLDL”) and intermediate density lipoprotein (“IDL”), and (2) anassociated reduction in clearance or conversion of said lipoproteins;Ouguerram et al., 2004 Arterioscler. Thromb. Vasc. Biol. 24:1448-1453.

Accordingly, there can be no doubt that PCSK9 plays a role in theregulation of LDL. The expression or upregulation of PCSK9 is associatedwith increased plasma levels of LDL cholesterol, and the correspondinginhibition or lack of expression of PCSK9 is associated with reduced LDLcholesterol plasma levels. Decreased levels of LDL cholesterolassociated with sequence variations in PCSK9 have been found to conferprotection against coronary heart disease; Cohen, 2006 N. Engl. J. Med.354:1264-1272.

The identification of compounds and/or agents effective in the treatmentof cardiovascular affliction is highly desirable. In clinical trials,reductions in LDL cholesterol levels have been directly related to therate of coronary events; Law et al., 2003 BMJ 326:1423-1427. Morerecently, the moderate lifelong reduction in plasma LDL cholesterollevels was found to correlate with a substantial reduction in theincidence of coronary events; Cohen et al, supra. This was the case evenin populations with a high prevalence of non-lipid-relatedcardiovascular risk factors; supra. Accordingly, there is great benefitto be reaped from the managed control of LDL cholesterol levels.

The present invention advances these interests by providing antagonistsof PCSK9 of use for inhibiting the activities of PCSK9 and thecorresponding role PCSK9 plays in various therapeutic conditions.

SUMMARY OF THE INVENTION

The present invention relates to antagonists of PCSK9 and, in particularembodiments, those antagonists that inhibit both human and murine PCSK9and those exhibiting preferential targeting of processed PCSK9. Broadly,protein-specific antagonists of PCSK9 (or “PCSK9-specific antagonists”as referred to herein) are PCSK9 protein binding molecules or moleculeseffective in the selective binding of PCSK9 and inhibition of PCSK9function. These molecules are of import in the treatment of conditionsassociated with or impacted by PCSK9 function, including, but notlimited to hypercholesterolemia, coronary heart disease, metabolicsyndrome, acute coronary syndrome and related conditions. PCSK9-specificantagonists are characterized by selective recognition and binding toPCSK9. PCSK9-specific antagonists do not show significant binding toproteins other than PCSK9, other than in those specific instances wherethe antagonist is supplemented or designed to confer an additional,distinct specificity to the PCSK9-specific binding component.

PCSK9-specific antagonists forming particular embodiments hereofcomprise (a) a heavy chain variable region comprising a CDR3 domaincomprising SEQ ID NO: 17 or an equivalent of SEQ ID NO: 17, saidequivalent characterized as having one or more conservative amino acidsubstitutions in the CDR3 domain; and/or (b) a light chain variableregion comprising a CDR3 domain comprising SEQ ID NO: 7 or an equivalentof SEQ ID NO: 7, said equivalent characterized as having one or moreconservative amino acid substitutions in the CDR3 domain. In specificembodiments, PCSK9-specific antagonists bind to human and/or murinePCSK9 with a K_(D) of 1.2×10⁻⁶ M or less. In more specific embodiments,PCSK9-specific antagonists bind to human and/or murine PCSK9 with aK_(D) of 1×10⁻⁷ M or less. In additional embodiments, PCSK9-specificantagonists bind to human and/or murine PCSK9 with a K_(D) of 1×10⁻⁸ Mor less. In further embodiments, PCSK9-specific antagonists bind tohuman and/or murine PCSK9 with a K_(D) of 5×10⁻⁹ M or less, or of 1×10⁻⁹M or less. In select embodiments, PCSK9-specific antagonists bind tohuman and/or murine PCSK9 with a K_(D) of 1×10⁻¹⁰ M or less, a K_(D) of1×10⁻¹1 M or less, or a K_(D) of 1×10⁻¹² M or less. In specificembodiments, PCSK9-specific antagonists do not bind proteins other thanPCSK9 at the above levels indicated for binding to PCSK9.

Particular embodiments of the present invention include PCSK9-specificantagonists which exhibit binding to PCSK9 at one of the aboveprescribed levels and compete for binding to PCSK9 with 1D05 antibodymolecules. 1D05 antibody molecules form important PCSK9-specificantagonists hereof. 1D05 antibody molecules are characterized ascomprising a (i) heavy chain variable region (“VH”) comprising SEQ IDNO: 11; and (ii) a light chain variable region (“VL”) comprising SEQ IDNO: 27. Said VH and VL regions comprise the full complement of disclosedCDRs 1, 2 and 3 for the VH (SEQ ID NOs: 13, 15 and 17) and VL regions(SEQ ID NOs: 3, 5 and 7), respectively. Examples of 1D05 antibodymolecules include without limitation: (i) a Fab which comprises a lightchain comprising SEQ ID NO: 1 and an Fd chain comprising amino acids1-233 of SEQ ID NO: 9 (or SEQ ID NO: 9); and (ii) a full length antibodymolecule which comprises a light chain comprising SEQ ID NO: 26 and aheavy chain comprising SEQ ID NO: 25.

PCSK9-specific antagonists are effective in counteractingPCSK9-dependent inhibition of cellular LDL-uptake, and particularlyhuman and/or murine PCSK9-dependent inhibition of cellular LDL uptake.Repeatedly, PCSK9-specific antagonist 1D05 has demonstrateddose-dependent inhibition of the effects of PCSK9 on LDL uptake.Accordingly, the disclosed PCSK9-specific antagonists are of import forlowering plasma LDL cholesterol levels. The disclosed antagonists alsohave utility for various diagnostic purposes, including the detectionand quantification of PCSK9. Select 1D05 antagonists are, in particular,useful because of their cross-reactivity with both human and murinePCSK9. This quality enables particular 1D05 antagonists to be studiedpharmacologically in murine models without having to ensure that themice express human PCSK9. In such experiments, the murine model issufficiently representative of the native activity of the targetedprotein and the antagonist's inhibition thereof.

In specific embodiments, the present invention encompassesPCSK9-specific antagonists. In particular embodiments, the presentinvention encompasses antibody molecules comprising disclosed heavyand/or light chain variable regions, equivalents of said regions havingone or more conservative amino acid substitutions, and homologs thereof.Select embodiments comprise isolated PCSK9-specific antagonists thatcomprise the disclosed CDR domains or sets of the heavy and/or lightchain CDR domains, and equivalents of such domains characterized ashaving one or more conservative amino acid substitutions. As will beappreciated by those skilled in the art, fragments of PCSK9-specificantagonists that retain the ability to antagonize PCSK9 may be insertedinto various frameworks; see, e.g., U.S. Pat. No. 6,818,418 andreferences contained therein, the collective disclosures of which areincorporated herein by reference, which discuss various scaffolds whichmay be used to display antibody loops previously selected on the basisof antigen binding. In the alternative, genes encoding for VL and VH maybe joined, using recombinant methods, for example using a syntheticlinker that enables them to be made as a single protein chain in whichthe VL and VH regions pair to form monovalent molecules, otherwise knownas single chain Fvs (“ScFVs”); see, e.g., Bird et al., 1988 Science 242:423-426, and Huston et al., 1988 Proc. Natl. Acad. Sci. USA85:5879-5883, the disclosures of which are incorporated herein byreference.

PCSK-9 specific antagonists and fragments may be in the form of variousnon-antibody-based scaffolds, including but not limited to avimers(Avidia); DARPins (Molecular Partners); Adnectins (Adnexus), Anticalins(Pieris) and Affibodies (Affibody). The use of alternative scaffolds forprotein binding is well appreciated in the scientific literature, see,e.g., Binz & Plückthun, 2005 Curr. Opin. Biotech. 16:1-11; thedisclosure of which is incorporated herein by reference. Accordingly,non-antibody-based scaffolds or antagonist molecules comprising (i) thedisclosed heavy and/or light chain variable region CDR3 sequences (SEQID NOs: 17 and 7, respectively), (ii) the disclosed heavy chain variableCDR1, CDR2 and CDR3 sequences or the disclosed light chain variableCDR1, CDR2 and CDR3 sequences: CDR1 (SEQ ID NOs: 13 and 3,respectively), CDR2 (SEQ ID NOs: 15 and 5, respectively) and CDR3 (SEQID NOs; 17 and 7, respectively, (iii) the full complement (SEQ ID NOs;13, 15, 17, 3, 5 and 7) of disclosed heavy and light chain CDRs within avariable region framework of a human heavy and/or light chain sequence,respectively, or (iv) the disclosed heavy and/or light chain variableregions SEQ ID NO: 11 and/or SEQ ID NO: 27 form important embodiments ofthe present invention, where such scaffolds or antagonist moleculesexhibit selectivity for PCSK9 and counteract PCSK9-dependent inhibitionof cellular LDL-uptake.

In another aspect, the present invention provides nucleic acid encodingthe disclosed PCSK9-specific antagonists and, in particular embodiments,PCSK9-specific antagonists which comprise the disclosed heavy and lightchains, the disclosed variable heavy and light regions and selectcomponents thereof (including CDRs 1, 2 and/or 3), particularly thedisclosed respective CDR3 regions. In another aspect, the presentinvention provides vectors comprising said nucleic acid. The presentinvention, additionally, provides isolated cell(s) comprising nucleicacid encoding disclosed PCSK9-specific antagonists. In another aspect,the present invention provides isolated cell(s) comprising a polypeptideor vector of the present invention.

The present invention provides methods for making PCSK9-specificantagonists disclosed herein including but not limited to antibodies,antigen binding fragments, derivatives, chimeric molecules, fusions ofany of the foregoing with another polypeptide, or alternativestructures/compositions capable of specifically binding PCSK9 whichcomprise the disclosed sequences. The methods comprise: (i) incubating acell comprising nucleic acid encoding the PCSK9-specific antagonist(s),or which comprises individual nucleic acids encoding one or morecomponents thereof, said nucleic acids which, when expressed,collectively produce the antagonist(s), under conditions that allow forthe expression and/or assembly of the PCSK9-specific antagonist(s), and(ii) isolating said antagonist(s) from the cell. One of skill in the artcan obtain PCSK9-specific antagonists disclosed herein using standardrecombinant DNA techniques as well.

The present invention provides a method for antagonizing the activity orfunction of PCSK9 or a noted effect of PCSK9 which comprises contactinga cell, population of cells, or tissue sample of interest expressingPCSK9 (or treated with or having therein human or murine PCSK9) with aPCSK9-specific antagonist disclosed herein under conditions that allowsaid antagonist to bind to PCSK9. Specific embodiments of the presentinvention include such methods wherein the cell is a human or murinecell. Additional embodiments are wherein the cell expresses human ormurine-derived PCSK9.

In another aspect, the present invention provides a method forantagonizing the activity or function of PCSK9 or a noted effect ofPCSK9 in a subject exhibiting a condition associated with PCSK9activity, or a condition where the functioning of PCSK9 iscontraindicated for a particular subject, which comprises administeringto the subject a therapeutically effective amount of a PCSK9-specificantagonist of the present invention in a pharmaceutical or othercomposition.

The present invention, thus, encompasses a method of treating acondition associated with PCSK9 activity, or a condition wherein thefunctioning of PCSK9 is contraindicated for a particular subject, whichcomprises administering to the subject a therapeutically effectiveamount of a PCSK9-specific antagonist of the present invention in apharmaceutical or other composition. In select embodiments, thecondition is hypercholesterolemia, coronary heart disease, metabolicsyndrome, acute coronary syndrome or related conditions.

In specific embodiments, the present invention encompasses a method ofadministering a disclosed PCSK9-specific antagonist to a subject whichcomprises delivering a therapeutically effective amount of apharmaceutical or other composition comprising a PCSK9-specificantagonist as disclosed herein.

In another aspect, the present invention provides a pharmaceuticalcomposition or other composition comprising a PCSK9-specific antagonistof the invention characterized as comprising a pharmaceuticallyacceptable carrier including but not limited to an excipient, diluent,stabilizer, buffer, or alternative designed to facilitate administrationof the antagonist in the desired amount to the treated individual.

The following table offers a generalized outline of the sequencesdiscussed in the present application. The Sequence Listing including allnotations, sequences and features forms as express part of thedisclosure hereof:

TABLE 1 SEQ ID NO: DESCRIPTION SEQ ID NO: 1 LIGHT CHAIN (“LC”); 1D05 SEQID NO: 2 LIGHT CHAIN (“LC”) NUCLEIC ACID; 1D05 SEQ ID NO: 3 VL CDR1;1D05 SEQ ID NO: 4 VL CDR1 NUCLEIC ACID; 1D05 SEQ ID NO: 5 VL CDR2; 1D05SEQ ID NO: 6 VL CDR2 NUCLEIC ACID; 1D05 SEQ ID NO: 7 VL CDR3; 1D05 SEQID NO: 8 VL CDR3 NUCLEIC ACID; 1D05 SEQ ID NO: 9 Fd CHAIN inclusive oflinkers and tags; 1D05 SEQ ID NO: 10 Fd CHAIN NUCLEIC ACID; 1D05 SEQ IDNO: 11 VH; 1D05 SEQ ID NO: 12 VH NUCLEIC ACID; 1D05 SEQ ID NO: 13 VHCDR1; 1D05 SEQ ID NO: 14 VH CDR1 NUCLEIC ACID; 1D05 SEQ ID NO: 15 VHCDR2; 1D05 SEQ ID NO: 16 VH CDR2 NUCLEIC ACID; 1D05 SEQ ID NO: 17 VHCDR3; 1D05 SEQ ID NO: 18 VH CDR3 NUCLEIC ACID; 1D05 SEQ ID NO: 19FRAGMENT OF PROCESSING SITE SEQ ID NO: 20 FRAGMENT OF PROCESSING SITESEQ ID NO: 21 Constant domain of IgG1 SEQ ID NO: 22 Constant domain ofIgG2 SEQ ID NO: 23 Constant domain of IgG4 SEQ ID NO: 24 Constant domainof IgG2m4 SEQ ID NO: 25 1D05 IgG2m4 Heavy Chain (“HC”) SEQ ID NO: 261D05 IgG Light (Kappa) Chain SEQ ID NO: 27 VL; 1D05 SEQ ID NO: 28 VLNUCLEIC ACID; 1D05 SEQ ID NO: 29 1D05 IgG2m4 HC NUCLEIC ACID SEQ ID NO:30 1D05 IgG LC NUCLEIC ACID SEQ ID NO: 31 1D05 IgG2m4 HC PLASMID SEQ IDNO: 32 1D05 IgG LC PLASMID SEQ ID NO: 33 PRIMER SEQ ID NO: 34 PRIMER SEQID NO: 35 PRIMER SEQ ID NO: 36 PRIMER SEQ ID NO: 37 1D05 EPITOPE DOMAINSEQ ID NO: 38 PORTION OF PCSK9 SEQUENCE IN FIGURE SEQ ID NO: 39 HUMANEPITOPE AREA SEQ ID NO: 40 CONSENSUS SEQUENCE SEQ ID NO: 41 MURINEEPITOPE AREA SEQ ID NO: 42 SECONDARY FOOTPRINT EPITOPE SEQ ID NO: 431D05 Variant VH CDR1 Sequence SEQ ID NO: 44 1D05 Variant VH CDR2Sequence SEQ ID NO: 45 1D05 Variant VH CDR3 Sequence SEQ ID NO: 46 1D05Variant VL CDR1 Sequence SEQ ID NO: 47 1D05 Variant VL CDR2 Sequence SEQID NO: 48 1D05 Variant VL CDR3 Sequence SEQ ID NO: 49 VL; 1D05 VariantSequence SEQ ID NO: 50 VH; 1D05 Variant Sequence SEQ ID NO: 51 VH; 1D05Variant Sequence H32Y SEQ ID NO: 52 VH; 1D05 Variant Sequence M48A SEQID NO: 53 VH; 1D05 Variant Sequence M48L SEQ ID NO: 54 VH; 1D05 VariantSequence H99Y SEQ ID NO: 55 VH; 1D05 Variant Sequence M48L/M109L/M115LSEQ ID NO: 56 VH; 1D05 Variant Sequence M48V SEQ ID NO: 57 Vl; 1D05Variant Sequence N50D SEQ ID NO: 58 Vl; 1D05 Variant Sequence N50Q SEQID NO: 59 Vl; 1D05 Variant Sequence N50T SEQ ID NO: 60 Vl; 1D05 VariantSequence N50Y

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates Fab expression vector pMORPH_x9_MH encoding themPCSK9 2CX1D05 Fab heavy and light chains.

FIG. 2 illustrates the activity of 1D05 in a PCSK9-LDLR interactionTR-FRET assay. Both the Fab and IgG of 1D05 are potent and inhibit theinteraction fully. For the experiment, [AF647-PCSK9]=10 nM, [Eu-sLDLR]˜1.5 nM (20000 counts at FI₆₂₀ nm).

FIGS. 3A-3D illustrate (i) 1D05 (Fab)'s dose-dependent inhibition ofmurine PCSK9-dependent loss of cellular LDL-uptake (FIG. 3A); (ii) 1D05(IgG)'s dose-dependent inhibition of murine PCSK9-dependent loss ofcellular LDL-uptake (FIG. 3B); (iii) 1D05 (Fab)'s dose-dependentinhibition of human PCSK9-dependent loss of cellular LDL-uptake (FIG.3C); and (iv) 1D05 (IgG)'s dose-dependent inhibition of humanPSCK9-dependent loss of cellular LDL-uptake (FIG. 3D). 1D05 clearlycross-reacts with both human and mouse PCSK9. FIGS. 3A-3D have twocontrols: (i) a cell only control, showing the basal level of cellularLDL uptake, and (ii) a cell+PCSK9 (5 μg/ml) control which shows thelevel of PCSK9-dependent loss of LDL-uptake. The titration experimentswhich contain 1D05 and PCSK9 were done at a fixed concentration of PCSK9(5 μg/ml) and increasing concentrations of 1D05 shown in the graphs. Asshown, 1D05 can inhibit the effect of PCSK9 on cellular LDL uptake.IC₅₀s for 1D05 (Fab) are 97 and 144 nM for mouse and human PCSK9protein, respectively. IC₅₀s for 1D05 (IgG) are 85 and 79 nM for mouseand human PCSK9 protein, respectively.

FIGS. 4A and 4B illustrate inhibition of PCSK9 internalization by 1D05(Fab and IgG, respectively) and restoration of LDL uptake. HepG2 cellswere plated and AlexaFluor-labeled PCSK9 and LDL were then added tocells and incubated at 37° C. for 4 hrs. Following incubation, theamount of PCSK9 or LDL internalized by cells was determined usingcofocal microscopy. Controls included the addition of cells alone (Notreatment), and only AF-labeled PCSK9 in addition to 50× (250 μg/ml)unlabeled PCSK9 (50× Cold Wt). In addition to 5 μg/ml wild-typeAF-labeled PCSK9 and 10 μg/ml AF-labeled LDL, increasing amounts ofeither the 1D05 Fab (panel A) or IGG (panel B) were added, resulting insubsequent inhibition of PCSK9 internalization into cells and a recoveryof LDL uptake. Together, these studies demonstrate that both the Fab andIgG prevent PCSK9 internalization into cells.

FIG. 5 illustrates the LDL levels for each mouse represented by a set ofconnected symbols; the change in LDL (postbleed-prebleed) being shown asan average for each treatment group (Δ mg/dL). Treatment with PBS had noeffect on LDL measurements (−4 mg/dL, 5% reduction). In contrast, serumLDL was reduced 20% with 1D05 whole IgG (−19 mg/dL) and 34% with Fabfragments of 1D05 (−24 mg/dL).

FIG. 6 illustrates a sequence comparison of the constant domains of IgG1(SEQ ID NO: 21; Fc domain of which is represented by residues 110-130 ofSEQ ID NO: 21), IgG2 (SEQ ID NO: 22, Fc domain of which is representedby residues 107-326 of SEQ ID NO: 22), IgG4 (SEQ ID NO: 23; Fc domain ofwhich is represented by residues 107-327 of SEQ ID NO: 23) and IgG2m4(SEQ ID NO: 24; Fc domain of which is represented by residues 107-326 ofSEQ ID NO: 24) isotypes.

FIGS. 7A and 7B illustrate peptide fragments originated by limitedproteolysis of a) wt-PCSK9 and b) 1D05/wt-PCSK9 complex with AspN for 5minutes. The star in FIG. 7A highlights the peptide fragment present inthe wt-PCSK9 spectrum which is not detected in the 1D05/wt-PCSK9spectrum. The aspartic acid residue 169 is hence protected in thecomplex.

FIGS. 8A-8H illustrate peptide fragments originated by limitedproteolysis of wt-PCSK9 (FIGS. 8A-8D) and 1D05/wt-PCSK9 complex (FIGS.8E-8H) with GluC for 15 minutes.

FIGS. 9A-9D illustrate peptide fragments originated by limitedproteolysis with Trypsin of a) wt-PCSK9 and b) 1D05/wt-PCSK9 complex for5 minutes. FIGS. 9C-D illustrate the zoom views of the same spectra,respectively. The stars in FIGS. 9A and 9C highlight the peptidefragments present in the wt-PCSK9 spectrum which are not detected in the1D05/wt-PCSK9 spectrum.

FIG. 10 illustrates residues of the primary sequence of the wt-PCSK9catalytic domain involved in binding with 1D05 neutralizing Fab. Thepeptide fragments of wt-PCSK9 protected in limited proteolysisexperiments by 1D05 binding are boxed.

FIG. 11 illustrates peptides of the wt-PCSK9 catalytic domain involvedin binding with 1D05 neutralizing Fab. The peptides of wt-PCSK9protected in limited proteolysis experiments by 1D05 binding aredepicted in the segments between R194 and R199, and A168 and R165 in thezoom view of the structure of wt-PCSK9.

FIGS. 12A and 12B illustrate sequence alignment between the identifiedgeneral epitope areas of human (SEQ ID NO: 39) and murine (SEQ ID NO:41) PCSK9. A consensus sequence (SEQ ID NO: 40) is provided as thesecond sequence of FIG. 12A. As evident in the Figures, the residuesincluded in the protected peptide 194-199 are conserved among the twospecies while the residues in peptide 165-169 are not.

FIG. 13 illustrates an analysis of 1D05 and a distinct antibody 1B20 ina PCSK9-1D05 interaction TR-FRET assay. Both Fabs are potent and inhibitthe interaction fully. For this experiment, [AF647-α-V5]=10 nM,[PCSK9]=10 nM, and [Eu(8044)-1D05(IgG)] ˜1.5 nM (˜18000 counts at F1620nm).

FIGS. 14A-D illustrates that 1B20 is a full inhibitor of PCSK9 functionin the Exopolar assay.

FIG. 15 illustrates 1D05 lowering LDL-C by ˜50% in rhesus at 3 mpk.Plotted are % LDL changes in serum at the different time points tested,post a single IV dose of antibody treatment.

FIG. 16 illustrates the pharmacokinetic profile of 1 D05 at the doselevels shown. Plotted are the serum drug (1D05) levels at time pointstested following a single IV dose of antibody. The half-life of 1D05 is77 hr.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to antagonists of PCSK9 and, in particularembodiments, those antagonists that inhibit both human and murine PCSK9and those that preferentially target processed PCSK9. Protein-specificantagonists of PCSK9 (or “PCSK9-specific antagonists”) in accordanceherewith are effective in the selective binding to and inhibition ofPCSK9 function and, thus, are of import in the treatment of conditionsassociated with or impacted by PCSK9 function, including, but notlimited to, hypercholesterolemia, coronary heart disease, metabolicsyndrome, acute coronary syndrome and related conditions. Use of theterm “antagonist” refers to the fact that the subject molecule canantagonize the functioning of PCSK9. Use of the term “antagonizing” orderivatives thereof refers to the act of opposing, counteracting,inhibiting, neutralizing or curtailing one or more functions of PCSK9.Reference herein to PCSK9 function or PCSK9 activity refers to anyfunction or activity that is driven by, requires, or is exacerbated orenhanced by PCSK9. PCSK9-specific antagonists as described herein haveproven to be effective for counteracting human and/or murinePCSK9-dependent inhibition of cellular LDL-uptake.

One important embodiment hereof relates to 1D05 antibody molecules. Such1D05 antibody molecules are characterized as comprising a (i) heavychain variable region (“VH”) comprising SEQ ID NO: 11; and (ii) a lightchain variable region (“VL”) comprising SEQ ID NO: 27. Said VH and VLregions comprise the full complement of disclosed CDRs 1, 2 and 3 forthe VH (SEQ ID NOs: 13, 15 and 17) and VL regions (SEQ ID NOs: 3, 5 and7), respectively. Examples of 1D05 antibody molecules include withoutlimitation: (i) a Fab which comprises a light chain comprising SEQ IDNO: 1 and an Fd chain comprising amino acids 1-233 of SEQ ID NO: 9 (orSEQ ID NO: 9); and (ii) a full length antibody molecule which comprisesa light chain comprising SEQ ID NO: 26 and a heavy chain comprising SEQID NO: 25. The select group of 1D05 antibodies demonstrate thatPCSK9-specific antagonists as disclosed herein effectively inhibit bothhuman and murine PCSK9 and may be studied pharmacologically in murinemodels absent the expression of human PCSK9.

The CDR definitions arrived at and disclosed herein were defined usingthe Morphosys software program Sequence Analysis Software (“SAS”).Applicants wish to note, however, that various other methods areavailable to delineate and define the start and end points of the CDRsequences, including but not limited to Kabat, 1991 Sequences ofProteins of Immunological Interest, 5^(th) edit, NIH Publication no.91-3242 U.S. Department of Health and Human Services; Clothia et al.,1987 J. Mol. Biol. 196:901-917; Clothia et al., 1989 Nature 342:877-883;Lefranc, 1997 Immunol. Today, 18:509; and Chen et al., 1999 J. Mol.Biol. 293:865-881. These and other methods have been reviewed and arewell within the realm of skills possessed by those in the art; see,e.g., Honegger & Plückthun, 2001 J. Mol. Biol. 309:657-670. While thecurrent inventors have employed the SAS software to define the CDRs, thepresent invention fully encompasses the different definitions around thesequences and the varying CDR delineations arrived at through use of anydifferent analysis software or methods. Said use and resulting CDRdefinitions based on the presently disclosed sequences is fully withinthe scope of the present disclosure and anticipated herein.

PCSK9-specific molecules also have utility for various diagnosticpurposes in the detection and quantification of PCSK9.

Disclosed PCSK9-specific antagonists are, furthermore, unique in thatselect embodiments have demonstrated a preferential recognition ofprocessed PCSK9, the active form of PCSK9.

PCSK9-specific antagonists as disclosed herein are desirable moleculesfor lowering plasma LDL cholesterol levels and are of utility for anyprimate, mammal or vertebrate of commercial or domestic veterinaryimportance. PCSK9-specific antagonists are of utility as well to inhibitthe activity of PCSK9 in any population of cells or tissues possessingthe LDL receptor. The utility of the disclosed antagonists is directlymeasurable by assays readily available to the skilled artisan. Means formeasuring LDL uptake are described in the literature; see, e.g., Barak &Webb, 1981 J. Cell Biol. 90:595-604, and Stephan & Yurachek, 1993 J.Lipid Res. 34:325330. In addition, means for measuring LDL cholesterolin plasma is well described in the literature; see, e.g., McNamara etal., 2006 Clinica Chimica Acta 369:158-167. The particular impact of thedisclosed antagonists on cellular LDL uptake may also be measuredthrough a method which comprises providing purified PCSK9 and labeledLDL particles to a cell sample; providing a PCSK9 antagonist to the cellsample; incubating said cell sample for a period of time sufficient toallow LDL particle uptake by the cells; quantifying the amount of labelincorporated into the cell; and identifying those antagonists thatresult in an increase in the amount of quantified label taken up by thecells as compared with that observed when PCSK9 is administered alone.An additional method for measuring the impact of the disclosedantagonists comprises providing purified PCSK9 and labeled LDL particlesto a cell sample; providing a PCSK9 antagonist to the cell sample;incubating said cell sample for a period of time sufficient to allow LDLparticle uptake by the cells; isolating cells of the cell sample byremoving the supernate; reducing non-specific association of labeled LDLparticles (whether to the plate, the cells, or anything other than theLDL receptor); lysing the cells; quantifying the amount of labelretained within the cell lysate; and identifying those antagonists thatresult in an increase in the amount of quantified label taken up by thecells as compared with that observed when PCSK9 is administered alone.Antagonists that result in an increase in the amount of quantified labelare PCSK9 antagonists.

Any type of cell bearing the LDL receptor can be employed in the abovemethods including, but not limited to HEK cells, HepG2 cells, and CHOcells. LDL particles derived from any source are of use in theabove-described assays. In particular assays, the LDL particles arefresh particles derived from blood. This can be accomplished by anymethod available to the skilled artisan including, but not limited to,the method of Havel et al., 1955 J. Clin. Invest. 34: 1345-1353. The LDLparticles may be labeled with fluorescence. The labeled LDL particlesmay have incorporated therein visible wavelength excited fluorophore3,3′-dioctadecylindocarbocyanine iodide (dil(3)) to form the highlyfluorescent LDL derivative dil(3)-LDL. Any label which enables theskilled artisan to detect LDL in the cellular lysate may be used. An LDLanalog may be used that would only become detectable (e.g., becomefluorescent or fluoresce at a different wavelength, etc.) whenmetabolized intracellularly or, for instance, if it were to becomeassociated with (or dissociated from) other molecules in the process ofbecoming internalized (e.g. a FRET assay, in which an LDL analog wouldbecome associated with a secondary fluor, or else be dissociated from aquencher). Any means available in the art for detecting internalizationof labeled LDL particles can be employed. The incubation time for theLDL particles and PCSK9 with the cells is an amount of time sufficientto allow LDL particle uptake by the cells. This time may be within therange of 5 minutes to 360 minutes. The concentration of PCSK9 added tothe cells may be in the range of 1 nM to 5 μM and, in specific methods,be in the range of 0.1 nM to 3 μM. One specific means by which theskilled artisan can determine a range of concentrations for a particularPCSK9 protein is to develop a dose response curve in the LDL-uptakeassay. A concentration of PCSK9 can be selected that promotes close tomaximal loss of LDL-uptake and is still in the linear range of the doseresponse curve. Typically, this concentration is ˜5 times the EC-50 ofthe protein extracted from the dose response curve. The concentrationscan vary by protein.

Broadly, PCSK9-specific antagonists as defined herein selectivelyrecognize and specifically bind to PCSK9. An antibody is typically saidto specifically bind an antigen when the dissociation constant is ≦1 μM,preferably ≦100 nM and most preferably ≦10 nM. Use of the terms“selective” or “specific” herein, further, refers to the fact that thedisclosed antagonists do not show significant binding to proteins otherthan PSCK9, except in those specific instances where the antagonist issupplemented or designed to confer an additional, distinct specificityto the PCSK9-specific binding portion (as, for example, in bispecific orbifunctional molecules where the molecule is designed to bind twomolecules or effect two functions, at least one of which is tospecifically bind PCSK9). In specific embodiments, PCSK9-specificantagonists bind to human and/or murine PCSK9 with a K_(D) of 1.2×10⁻⁶ Mor less. In more specific embodiments, PCSK9-specific antagonists bindto human and/or murine PCSK9 with a K_(D) of 5×10⁻⁷ M or less, of 2×10⁻⁷M or less, or of 1×10⁻⁷ M or less. In additional embodiments,PCSK9-specific antagonists bind to human and/or murine PCSK9 with aK_(D) of 1×10⁻⁸ M or less. In further embodiments, PCSK9-specificantagonists bind to human and/or murine PCSK9 with a K_(D) of 5×10⁻⁹ Mor less, or of 1×10⁻⁹ M or less. In select embodiments, PCSK9-specificantagonists bind to human and/or murine PCSK9 with a K_(D) of 1×10⁻¹⁰ Mor less, a K_(D) of 1×10⁻¹¹ M or less, or a K_(D) of 1×10⁻¹² M or less.In specific embodiments, PCSK9-specific antagonists do not bind proteinsother than PCSK9 at the above K_(D)s. K_(D) refers to the dissociationconstant obtained from the ratio of K_(d) (the dissociation rate of aparticular binding molecule-target protein interaction) to K_(a) (theassociation rate of the particular binding molecule-target proteininteraction), or K_(d)/K_(a) which is expressed as a molar concentration(M). K_(D) values can be determined using methods well established inthe art. A preferred method for determining the K_(D) of a bindingmolecule is by using surface plasmon resonance, for example employing abiosensor system such as a Biacore™ (GE Healthcare Life Sciences)system.

PCSK9-specific antagonists disclosed herein have been shown todose-dependently inhibit human and/or murine PCSK9 dependent effects onLDL uptake. Accordingly, PCSK9-specific antagonists as disclosed hereinare characterized by their ability to counteract PCSK9-dependentinhibition of LDL uptake into cells. This uptake of LDL into cells bythe LDL receptor is referred to herein as “cellular LDL uptake”. Inspecific embodiments, PCSK9-specific antagonists counteract orantagonize human and/or murine PCSK9-dependent inhibition of LDL uptakeinto cells, exhibiting an IC₅₀ of less than 1.0×10⁻⁶ M, or, in order ofpreference, less than 1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ Mand 1×10⁻¹² M. The extent of inhibition by any PCSK9-specific antagonistmay be measured quantitatively in statistical comparison to a control,or via any alternative method available in the art for assessing anegative effect on, or inhibition of, PCSK9 function (i.e., any methodcapable of assessing antagonism of PCSK9 function). In specificembodiments, the inhibition is at least about 10% inhibition. In otherembodiments, the inhibition is at least 20%, 30%, 40%, 50%, 60%, 70,%,80%, 90%, or 95%. Accordingly, PCSK9-specific antagonists capable ofeffecting these levels of inhibition of PCSK9 function form particularembodiments hereof.

A PCSK9-specific antagonist in accordance herewith can be any bindingmolecule that specifically binds human and/or murine PCSK9 proteinincluding, but not limited to, antibody molecules as defined below, anyPCSK9-specific binding structure, any polypeptide or nucleic acidstructure that specifically binds PCSK9, and any of the foregoingincorporated into various protein scaffolds; including but not limitedto, various non-antibody-based scaffolds, and various structures capableof affording or allowing for selective binding to PCSK9 including butnot limited to small modular immunopharmaceuticals (or “SMIPs”; see,Haan & Maggos, 2004 Biocentury Jan. 26); Immunity proteins (see, e.g.,Chak et al., 1996 Proc. Natl. Acad. Sci. USA 93:6437-6442); cytochromeb562 (see Ku and Schultz, 1995 Proc. Natl. Acad. Sci. USA 92:6552-6556);the peptide α2p8 (see Barthe et al., 2000 Protein Sci. 9:942-955);avimers (Avidia; see Silverman et al., 2005 Nat. Biotechnol.23:1556-1561); DARPins (Molecular Partners; see Binz et al., 2003 J.Mol. Biol. 332:489-503; and Forrer et al., 2003 FEBS Lett. 539:2-6);Tetranectins (see, Kastrup et al., 1998 Acta. Crystallogr. D. Biol.Crystallogr. 54:757-766); Adnectins (Adnexus; see, Xu et al., 2002 Chem.Biol. 9:933-942), Anticalins (Pieris; see Vogt & Skerra, 2004Chemobiochem. 5:191-199; Beste et al., 1999 Proc. Natl. Acad. Sci. USA96:1898-1903; Lamla & Erdmann, 2003 J. Mol. Biol. 329:381-388; and Lamla& Erdmann, 2004 Protein Expr. Purif. 33:39-47); A-domain proteins (seeNorth & Blacklow, 1999 Biochemistry 38:3926-3935), Lipocalins (seeSchlehuber & Skerra, 2005 Drug Discov. Today 10:23-33); Repeat-motifproteins such as Ankyrin repeat proteins (see Sedgwick & Smerdon, 1999Trends Biochem. Sci. 24:311-316; Mosavi et al., 2002 Proc. Natl. Acad.Sci. USA 99:16029-16034; and Binz et al., 2004 Nat. Biotechnol22:575-582); Insect Defensin A (see Zhao et al., 2004 Peptides25:629-635); Kunitz domains (see Roberts et al., 1992 Proc. Natl. Acad.Sci. USA 89:2429-2433; Roberts et al., 1992 Gene 121:9-15; Dennis &Lazarus, 1994 J. Biol. Chem. 269:22129-22136; and Dennis & Lazarus, 1994J. Biol. Chem. 269:22137-22144); PDZ-Domains (see Schneider et al., 1999Nat. Biotechnol. 17:170-175); Scorpion toxins such as Charybdotoxin (seeVita et al., 1998 Biopolymers 47:93-100); 10^(th) fibronectin type IIIdomain (or 10Fn3; see Koide et al., 1998 J. Mol. Biol. 284:1141-1151,and Xu et al., 2002 Chem. Biol. 9:933-942); CTLA-4 (extracellulardomain; see Nuttall et al., 1999 Proteins 36:217-227; and Irving et al.,2001 J. Immunol. Methods 248:31-45); Knottins (see Souriau et al., 2005Biochemistry 44:7143-7155 and Lehtio et al., 2000 Proteins 41:316-322);Neocarzinostatin (see Heyd et al. 2003 Biochemistry 42:5674-5683);carbohydrate binding module 4-2 (CBM4-2; see Cicortas et al., 2004Protein Eng. Des. Sel. 17:213-221); Tendamistat (see McConnell & Hoess,1995 J. Mol. Biol. 250:460-470, and Li et al., 2003 Protein Eng.16:65-72); T cell receptor (see Holler et al., 2000 Proc. Natl. Acad.Sci. USA 97:5387-5392; Shusta et al., 2000 Nat. Biotechnol. 18:754-759;and Li et al., 2005 Nat. Biotechnol. 23:349-354); Affibodies (Affibody;see Nord et al., 1995 Protein Eng. 8:601-608; Nord et al., 1997 Nat.Biotechnol. 15:772-777; Gunneriusson et al., 1999 Protein Eng.12:873-878); and other selective binding proteins or scaffoldsrecognized in the literature; see, e.g., Binz & Plückthun, 2005 Curr.Opin. Biotech. 16:1-11; Gill & Damle, 2006 Curr. Opin. Biotechnol.17:1-6; Hosse et al., 2006 Protein Science 15:14-27; Binz et al., 2005Nat. Biotechnol. 23:1257-1268; Hey et al., 2005 Trends in Biotechnol.23:514-522; Binz & Plückthun, 2005 Curr. Opin. Biotech. 16:459-469;Nygren & Skerra, 2004 J. Immunolog. Methods 290:3-28; Nygren & Uhlen,1997 Curr. Opin. Struct. Biol. 7:463-469; the disclosures of which areincorporated herein by reference. Antibodies and the use ofantigen-binding fragments is well defined and understood in theliterature. The use of alternative scaffolds for protein binding is wellappreciated in the scientific literature as well, see, e.g., Binz &Plückthun, 2005 Curr. Opin. Biotech. 16:1-11; Gill & Damle, 2006 Curr.Opin. Biotechnol. 17:1-6; Hosse et al., 2006 Protein Science 15:14-27;Binz et al., 2005 Nat. Biotechnol. 23:1257-1268; Hey et al., 2005 Trendsin Biotechnol. 23:514-522; Binz & Plückthun, 2005 Curr. Opin. Biotech.16:459-469; Nygren & Skerra, 2004 J. Immunolog. Methods 290:3-28; Nygren& Uhlen, 1997 Curr. Opin. Struct. Biol. 7:463-469; the disclosures ofwhich are incorporated herein by reference. Accordingly,non-antibody-based scaffolds or antagonist molecules in accordanceherewith exhibiting selectivity for PCSK9 that counteractPCSK9-dependent inhibition of cellular LDL-uptake form importantembodiments of the present invention. Aptamers (nucleic acid or peptidemolecules capable of selectively binding a target molecule) are onespecific example. They can be selected from random sequence pools oridentified from natural sources such as riboswitches. Peptide aptamers,nucleic acid aptamers (e.g., structured nucleic acid, including both DNAand RNA-based structures) and nucleic acid decoys can be effective forselectively binding and inhibiting proteins of interest; see, e.g.,Hoppe-Seyler & Butz, 2000 J. Mol. Med. 78:426-430; Bock et al., 1992Nature 355:564-566; Bunka & Stockley, 2006 Nat. Rev. Microbiol.4:588-596; Martell et al., 2002 Molec. Ther. 6:30-34; Jayasena, 1999Clin. Chem. 45:1628-1650; the disclosures of which are incorporatedherein by reference.

Importantly, the binding site (or epitope) for 1D05 on PCSK9 wasidentified through limited proteolysis and mass spectrometry (“LP-MS”).The limited proteolysis mass spectrometry analysis involved incubatingwild-type PCSK9 (“wt-PCSK9”) and a complex of wt-PCSK9 and 1D05 withendoproteinase enzymes of different specificity in carefully controlledconditions. Under such conditions, the endoproteases cleaved onlyexposed primary cleavage sites. The experiment was designed so that thebinding of 1D05 to wt-hPCSK9 masked surface residues normally exposed onwt-hPCSK9 not bound to the antibody. Such masked residues providedinsight into the binding domain of 1D05. Through such experiments, anovel neutralizing epitope conformational in nature and represented bypeptides RYRAD (SEQ ID NO: 42) AND REIEGR (SEQ ID NO: 37) wasidentified. This epitope falls within PCSK9's catalytic domain andprovides a novel target epitope for which to identify additionaleffective PCSK9 antagonists. Identification of additional PCSK9-specificantagonists binding this epitope is of significant interest given 1D05'sPCSK9-neutralizing activity.

One means of identifying antagonists and particularly antibodies thatbind to the identified 1D05 epitope or an overlapping epitope is througha competition or similar assay where the candidate antibody or bindingmolecule would have to out-compete 1D05 for the epitope. Competitiveantagonists encompassed herein are molecules that inhibit (i.e., preventor interfere with in comparison to a control) or reduce 1D05 binding byat least 50%, 60%, 70%, and 80% in order of increasing preference (evenmore preferably, at least 90% and, most preferably, at least 95%) at 1μM or less with 1D05 at or below its K_(D), and in particular thosemolecules that antagonize (i) PCSK9 binding to the LDL receptor, (ii)PCSK9 internalization into cells, or (iii) both PCSK9 binding to the LDLreceptor and PCSK9 internalization into cells. Competition betweenbinding members may be readily assayed in vitro for example using ELISAand/or by monitoring the interaction of the antibodies with PCSK9 insolution. The exact means for conducting the analysis is not critical.PCSK9 may be immobilized to a 96-well plate or may be placed in ahomogenous solution. In specific embodiments, the ability of unlabeledcandidate antibody(ies) to block the binding of labeled 1D05 can bemeasured using radioactive, enzyme or other labels. In the reverseassay, the ability of unlabeled antibodies to interfere with theinteraction of labeled 1D05 with PCSK9 wherein said 1D05 and PCSK9 arealready bound is determined. In specific embodiments, (i) PCSK9 iscontacted with labeled 1D05 (an antibody molecule which comprises a VLcomprising SEQ ID NO: 27 and a VH comprising. SEQ ID NO: 11); (ii) PCSK9is contacted with the candidate antibody or pool of antibodies; and(iii) antibodies capable of interrupting or preventing complexes betweenPCSK9 and 1D05 are identified. The readout in such an example is throughmeasurement of bound label. 1D05 and the candidate antibody(ies) may beadded in any order or at the same time. A specific assay that may be runis that of Example 13 where the activity of an antibody found to bind tothe same epitope domain as 1D05 is illustrated.

Antibodies identified as 1D05 competitors in the above or other suitableassays may be tested for the ability to antagonize or neutralize (i)PCSK9 binding to the LDL receptor; and/or (ii) PCSK9 internalizationinto cells. These parameters may be measured through the use of assayssimilar to that employed or described in the current specification. Inspecific embodiments, the inhibition demonstrated by the competingantibody is at least about 10% inhibition. In other embodiments, theinhibition is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

The present invention specifically encompasses PCSK9-specificantagonists and particularly monoclonal antibody molecules (and theircorresponding amino acid and nucleic acid sequences) that selectivelybind to the epitope identified for 1D05 or an overlapping epitopeinterfering with 1D05's binding to PCSK9. Critical residues for 1D05binding that were identified on the epitope of PCSK9 are those residuescorresponding to residues Arg194, Glu197 and Arg199 of human PCSK9. Thenarrow epitope comprising these amino acid residues is represented bySEQ ID NO: 37 and falls within the area of SEQ ID NO: 39 of human PCSK9and SEQ ID NO: 41 of murine PCSK9. A secondary footprint of the antibodyis represented by SEQ ID NO: 42. Monoclonal antibodies that specificallybind to the conformational epitope represented by SEQ ID NO: 37 and SEQID NO:42 or an overlapping epitope antagonize or neutralize (i) PCSK9binding to the LDL receptor; (ii) PCSK9 internalization into cells, or(iii) both. Accordingly, monoclonal antibodies that bind to an epitopeon PCSK9 which comprises and/or consists of: SEQ ID NO: 37, SEQ ID NO:39 or SEQ ID NO: 41 form important embodiments of the present invention.Specific embodiments of the present invention relate to monoclonalantibodies that recognize the following epitopes on PCSK9: SEQ ID NO: 37and SEQ ID NO: 42. A monoclonal antibody molecule in accordance herewithmay be an intact (complete or full length) antibody, a substantiallyintact antibody, or a portion or fragment of an antibody comprising anantigen-binding portion, e.g., a Fab fragment, Fab′ fragment or F(ab′)₂fragment of a murine antibody or of a chimeric antibody or of ahumanized antibody or of a human antibody. Monoclonal, as used herein,refers to a homogeneous or substantially homogeneous (or pure) antibodypopulation (i.e., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, morepreferably at least about 97% or 98%, or most preferably at least 99% ofthe antibodies in the population are identical and would compete in anELISA assay for the same antigen or epitope. In specific embodiments ofthe present invention, the present invention provides monoclonalantibodies that (i) compete for binding to PCSK9 with a 1D05 antibodymolecule, reducing 1D05 binding by at least 50% at 1 μM or less with1D05 at or below its K_(D), (ii) block PCSK9 binding to the LDLreceptor, (iii) inhibit PCSK9 internalization into the cell, and (iv)comprise a specific antigen-binding region, VH, VL, set of CDRs or heavyCDR3, heavy and/or light chain or any variant of these componentsdescribed herein. Additional embodiments provide PCSK9-specificantagonists including but not limited to monoclonal antibodies thatrecognize/bind to SEQ ID NO: 37, SEQ ID NO: 39 or SEQ ID NO: 41, whereinthe PCSK9-specific antagonists bind to human and/or murine PCSK9 with aK_(D) of 1.2×10⁻⁶ M or less, and wherein the PCSK9-specific antagonistcompetes with 1D05 for binding to PCSK9. In specific embodiments hereof,the PCSK9-specific antagonists are further defined by one or more of thefollowing qualities: they (i) reduce 1D05 binding by at least 50% at 1μM or less with 1D05 at or below its K_(D), (ii) block PCSK9 binding tothe LDL receptor, (iii) inhibit PCSK9 internalization into the cell,and/or (iv) comprise a specific antigen-binding region, VH, VL, set ofCDRs or heavy CDR3, heavy and/or light chain or any variant of thesecomponents described herein. In specific embodiments, the PCSK9-specificantagonists in accordance with the above comprise (i) the disclosedheavy and/or light chain variable region CDR3 sequences (SEQ ID NOs: 17and 7, respectively), (ii) the disclosed heavy and/or light chainvariable regions CDR1 (SEQ ID NOs: 13 and 3, respectively), CDR2 (SEQ IDNOs: 15 and 5, respectively) and CDR3 (SEQ ID NOs; 17 and 7,respectively, (iii) the full complement (SEQ ID NOs; 13, 15, 17, 3, 5and 7) of disclosed heavy and light chain CDRs within a variable regionframework of a human heavy and/or light chain sequence; (iv) thedisclosed VL and/or VH regions (SEQ ID NOs: 27 and 11, respectively);(v) the disclosed light and/or Fd chains (SEQ ID NO: 1 and amino acids1-233 of SEQ ID NO: 9 (or SEQ ID NO: 9)), or (vi) the disclosed lightand/or heavy chains (SEQ ID NOs: 26 and 25). In specific embodiments,the PCSK9-specific antagonists bind to/recognize both SEQ ID NOs: 37 andSEQ ID NO: 42.

In any of the above assays for identifying antibodies binding the sameor overlapping epitope region as 1D05, binding of the known binder(i.e., 1D05 antibody molecule known to bind residues Arg194, Glu197 andArg199 of SEQ ID NO: 37) as compared to the binding of the candidatebinder should be distinguishable. This can (but need not) beaccomplished through the use of labels on either or both molecules aswill be readily appreciated by the skilled artisan. Labels, as usedherein, refer to another molecule or agent incorporated into/affixed tothe antibody molecule. In one embodiment, the label is a detectablemarker, e.g., a radiolabeled amino acid or attachment to a polypeptideof biotinyl moieties that can be detected by marked avidin (e.g.,streptavidin containing a fluorescent marker or enzymatic activity thatcan be detected by optical or colorimetric methods). Various methods oflabeling polypeptides and glycoproteins are known in the art and may beused. Examples of labels for polypeptides include, but are not limitedto, the following: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N,35S, ⁹⁰Y, 99Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC,rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradishperoxidase, β-galactosidase, luciferase, alkaline phosphatase),chemiluminescent markers, biotinyl groups, predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags), magnetic agents, such as gadolinium chelates,toxins such as pertussis toxin, taxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance.

A 1D05 antibody used for the competition assays may be any antibodymolecule which is of the 1D05 description provided herein (i.e. anyantibody molecule selective for PCSK9 which comprises a VL comprisingSEQ ID NO: 27 and a VH comprising SEQ ID NO: 11). Examples of suchantibodies include without limitation (i) a Fab which comprises a lightchain comprising SEQ ID NO: 1 and an Fd chain comprising amino acids1-233 of SEQ ID NO: 9 (or SEQ ID NO: 9); (ii) a full length antibodymolecule which comprises a light chain comprising SEQ ID NO: 26 and aheavy chain comprising SEQ ID NO: 25.

Peptides or peptidomimetics based on the regions corresponding to SEQ IDNO: 39 or SEQ ID NO: 41 (and in select embodiments the areascorresponding to SEQ ID NO: 37 and SEQ ID NO: 42) should haveantagonistic properties by preventing the interaction of PCSK9 withLDLR. Importantly, peptides that comprise SEQ ID NO: 37 and SEQ ID NO:42 should generate neutralizing antibodies able to inhibit PCSK9 bindingto LDLR and/or inhibit PCSK9 internalization into cells.

In specific embodiments, peptides encompassed herein comprise SEQ ID NO:39 OR SEQ ID NO: 41. In select embodiments, the peptides comprise SEQ IDNO: 37 and are less than 50 amino acids. In certain embodiments, thepeptides comprise both SEQ ID NO: 37 and SEQ ID NO: 42 and are 40 aminoacids or less. In more specific embodiments, the peptides comprise SEQID NO: 37 and are less than 40 amino acids, less than 30 amino acids,less than 20 amino acids, or less than 10 amino acids.

Screening of peptides of the invention may be carried out utilizingcompetition assays as described above. If the peptide being testedcompetes with a 1D05 antibody molecule (i.e. any antibody moleculeselective for PCSK9 which comprises a VL comprising SEQ ID NO: 27 and aVH comprising SEQ ID NO: 11) as shown by a decrease in binding of such1D05 antibody molecule then it is likely that the peptide and 1D05 bindto the same, or a closely related, epitope. Still another way todetermine whether a peptide has the specificity of the 1D05 antibodymolecule is to pre-incubate the 1D05 antibody molecule with PCSK9 withwhich it is normally reactive, and then add the peptide being testedwith demonstrated specificity for PCSK9 to determine whether the peptideis inhibited in its ability to bind PCSK9. If the peptide being testedis inhibited then, in all likelihood, it has the same, or a functionallyequivalent, epitope and specificity as the 1D05 antibody molecule.

Using routine procedures as outlined throughout the instantspecification and well known to those of ordinary skill in the art, onecan then determine whether a peptide which binds to PCSK9 is useful bydetermining whether the peptide is blocks PCSK9 from binding to the LDLreceptor and/or prevents PCSK9 internalization into cells.

Expression and selection of any of the PCSK9-specific antagonistsdescribed in the present application may be achieved using suitabletechnologies including, but not limited to phage display (see, e.g.,International Application Number WO 92/01047, Kay et al., 1996 PhageDisplay of Peptides and Proteins: A Laboratory Manual, San Diego:Academic Press), yeast display, bacterial display, T7 display, andribosome display (see, e.g., Lowe & Jermutus, 2004 Curr. Pharm. Biotech.517-527).

Particular PCSK9-specific antagonists forming part of the presentinvention are antibody molecules or antibodies. “Antibody molecule” or“Antibody” as described herein refers to an immunoglobulin-derivedstructure with selective binding to human and/or murine PCSK9 including,but not limited to, a full length or whole antibody, an antigen bindingfragment (a fragment derived, physically or conceptually, from anantibody structure), a derivative of any of the foregoing, a fusion ofany of the foregoing with another polypeptide, or any alternativestructure/composition which incorporates any of the foregoing forpurposes of selectively binding to/inhibiting the function of PCSK9.

“Whole” antibodies or “full length” antibodies refer to proteins thatcomprise two heavy (H) and two light (L) chains inter-connected bydisulfide bonds which comprise: (1) in terms of the heavy chains, avariable region (abbreviated herein as “VH”) and a heavy chain constantregion which comprises three domains, C_(H1), C_(H2), and C_(H3); and(2) in terms of the light chains, a light chain variable region(abbreviated herein as “V_(L)”) and a light chain constant region whichcomprises one domain, C_(L).

Antibody fragments and, more specifically, antigen binding fragments aremolecules possessing an antibody variable region or segment thereof(which comprises one or more of the disclosed CDR 3 domains, heavyand/or light within framework regions of heavy and/or light chains, asappropriate), which confers selective binding to PCSK9, and particularlyhuman and/or murine PCSK9. Antibody fragments containing such anantibody variable region include, but are not limited to the followingantibody molecules: a Fab, a F(ab′)2, a Fd, a Fv, a scFv, bispecificantibody molecules (antibody molecules comprising a PCSK9-specificantibody or antigen binding fragment as disclosed herein linked to asecond functional moiety having a different binding specificity than theantibody, including, without limitation, another peptide or protein suchas an antibody, or receptor ligand), a bispecific single chain Fv dimer,an isolated CDR3, a minibody, a ‘scAb’, a dAb fragment, a diabody, atriabody, a tetrabody, a minibody, and artificial antibodies based uponprotein scaffolds, including but not limited to fibronectin type IIIpolypeptide antibodies (see, e.g., U.S. Pat. No. 6,703,199 andInternational Application Numbers WO 02/32925 and WO 00/34784) orcytochrome B; see, e.g., Nygren et al., 1997 Curr. Opinion Struct. Biol.7:463-469; the disclosures of which are incorporated herein byreference. The antibody portions or binding fragments may be natural, orpartly or wholly synthetically produced. Such antibody portions can beprepared by various means known by one of skill in the art, including,but not limited to, conventional techniques, such as papain or pepsindigestion.

The term “isolated” as used herein in reference to antibody molecules,PCSK9-specific antagonists in general, encoding nucleic acid or otherdescribes a property as it pertains to the disclosed PCSK9-specificantagonists, nucleic acid or other that makes them different from thatfound in nature. The difference can be, for example, that they are of adifferent purity than that found in nature, or that they are of adifferent structure or form part of a different structure than thatfound in nature. A structure not found in nature, for example, includesrecombinant human immunoglobulin structures including, but not limitedto, recombinant human immunoglobulin structures with optimized CDRs.Other examples of structures not found in nature are PCSK9-specificantagonists or nucleic acid substantially free of other cellularmaterial. Isolated PCSK9-specific antagonists are generally free ofother protein-specific antagonists having different proteinspecificities (i.e., possess an affinity for other than PCSK9).

In one particular aspect, the present invention provides isolatedPCSK9-specific antagonists which antagonize PCSK9 function. Inparticular embodiments, said PCSK9-specific antagonists inhibit humanand/or murine PCSK9's antagonism of cellular LDL uptake by interferingwith PCSK9 binding to the LDL receptor and resultant PCSK9 cellinternalization. Disclosed PCSK9-specific antagonists, thus, formdesirable molecules for lowering plasma LDL-cholesterol levels; see,e.g., Cohen et al., 2005 Nat. Genet 37:161-165 (wherein significantlylower plasma LDL cholesterol levels were noted in individualsheterozygous for a nonsense mutation in allele PCSK9); Rashid et al.,2005 Proc. Natl. Acad. Sci. USA 102:5374-5379 (wherein PCSK9-knockoutmice evidenced increased numbers of LDLRs in hepatocytes, acceleratedplasma LDL clearance, and significantly lower plasma cholesterollevels); and Cohen et al., 2006 N Engl. J. Med. 354:1264-1272 (whereinhumans heterozygous for mutated, loss of function, PCSK9 exhibited asignificant reduction in the long-term risk of developingatherosclerotic heart disease).

Through repeat experiments, 1D05 antibody molecules as disclosed hereindose-dependently inhibited the effects of both human and/or murine PCSK9on LDL uptake. In specific embodiments, the present invention, thus,encompasses PCSK9-specific antagonists and, in more specificembodiments, antibody molecules comprising the heavy and/or light chainvariable regions (SEQ ID NO: 11 and 27, respectively) contained withinthese 1D05 antibody molecules or the heavy and/or light chains, e.g.,amino acids 1-233 of SEQ ID NO: 9 (or SEQ ID NO: 9) and SEQ ID NO: 1,respectively, or SEQ ID NOs: 25 and 26, respectively, as well asequivalents (characterized as having one or more conservative amino acidsubstitutions that do not degrade the PCSK9-selective property of 1D05)or homologs thereof. Particular embodiments comprise isolatedPCSK9-specific antagonists that comprise the CDR domains disclosedherein or sets of heavy and/or light chain CDR domains disclosed herein,or equivalents thereof, characterized as having one or more conservativeamino acid substitutions.

Use of the terms “domain” or “region” herein simply refers to therespective portion of the antibody molecule wherein the sequence orsegment at issue will reside or, in the alternative, currently resides.

In specific embodiments, the present invention provides isolatedPCSK9-specific antagonists and, in more specific embodiments, antibodymolecules comprising a heavy chain variable region which comprises SEQID NO: 11; equivalents thereof characterized as having one or moreconservative amino acid substitutions, and homologs thereof. Thedisclosed antagonists should counteract or inhibit human and/or murinePCSK9-dependent inhibition of cellular LDL uptake. In specificembodiments, the present invention provides homologs of the disclosedantagonists characterized as being at least 90% identical over the heavychain variable region to SEQ ID NO: 11; said antagonists which inhibithuman and/or murine PCSK9-dependent inhibition of cellular LDL uptake byat least 10%.

In specific embodiments, the present invention provides isolatedPCSK9-specific antagonists and, in more specific embodiments, antibodymolecules comprising a light chain variable region which comprises SEQID NO: 27; equivalents thereof characterized as having one or moreconservative amino acid substitutions, and homologs thereof. Thedisclosed antagonists should counteract or inhibit human and/or murinePCSK9-dependent inhibition of cellular LDL uptake. In specificembodiments, the present invention provides homologs of the disclosedantagonists characterized as being at least 90% identical over the lightchain variable region to SEQ ID NO: 27; said antagonists which inhibithuman and/or murine PCSK9-dependent inhibition of cellular LDL uptake byat least 10%.

In specific embodiments, the present invention provides isolatedPCSK9-specific antibody molecules which comprise a heavy chain variableregion comprising SEQ ID NO: 11 and a light chain variable regioncomprising SEQ ID NO: 27; or equivalents thereof characterized as havingone or more conservative amino acid substitutions in the prescribedsequences. Specific embodiments are said antagonists which inhibit humanand/or murine PCSK9-dependent inhibition of cellular LDL uptake by atleast 10%. In specific embodiments, the present invention provideshomologs of the disclosed antagonists characterized as being at least90% identical over the heavy and light chain variable regions to SEQ IDNOs: 11 and 27, respectively; said antagonists which inhibit humanand/or murine PCSK9-dependent inhibition of cellular LDL uptake by atleast 10%.

In particular embodiments, the present invention provides isolatedPCSK9-specific antagonists and, in more specific embodiments, PCSK9antibody molecules that comprise variable heavy CDR3 sequence SEQ ID NO:17; and equivalents thereof characterized as having one or moreconservative amino acid substitutions; specific embodiments of whichinhibit human and/or murine PCSK9-dependent inhibition of cellular LDLuptake by at least 10%. Specific embodiments provide isolatedantagonists which additionally comprise in the heavy chain variableregion CDR1 and/or CDR2 sequences comprising SEQ ID NO: 13 and/or SEQ IDNO: 15, respectively; or equivalents thereof characterized as having oneor more conservative amino acid substitutions in any one ore more of theCDR sequences. In specific embodiments, the present invention provideshomologs of the disclosed antagonists characterized as being at least90% identical over the CDR3 sequences or within each of the CDR1, CDR2and CDR3 sequences to SEQ ID NO: 17 or SEQ ID NOs: 13, 15 and 17,respectively, as appropriate; said antagonists which inhibit humanand/or murine PCSK9-dependent inhibition of cellular LDL uptake by atleast 10%.

In particular embodiments, the present invention provides isolatedPCSK9-specific antagonists and, in more specific embodiments, antibodymolecules which comprise variable light CDR3 sequence which comprisesSEQ ID NO: 7; and equivalents thereof characterized as having one ormore conservative amino acid substitutions; specific embodiments ofwhich inhibit human and/or murine PCSK9-dependent inhibition of cellularLDL uptake by at least 10%. Specific embodiments provide isolatedantagonists which additionally comprise in the light chain variableregion CDR1 and/or CDR2 sequences comprising SEQ ID NO: 3 and/or SEQ IDNO: 5, respectively; or an equivalent thereof characterized as havingone or more conservative amino acid substitutions in any one or more ofthe CDR sequences. In specific embodiments, the present inventionprovides homologs of the disclosed antagonists characterized as being atleast 90% identical over the CDR3 sequences or within each of the CDR1,CDR2 and CDR3 sequences to SEQ ID NO: 7 or SEQ ID NOs: 3, 5 and 7,respectively, as appropriate; said antagonists which inhibit humanand/or murine PCSK9-dependent inhibition of cellular LDL uptake by atleast 10%.

In particular embodiments, the present invention provides isolatedPCSK9-specific antagonists and, in more specific embodiments, antibodymolecules which comprise heavy chain variable region CDR3 sequence andlight chain variable region CDR3 sequence comprising SEQ ID NOs: 17 and7, respectively; or equivalents thereof characterized as having one ormore conservative amino acid substitutions in any one or more of theCDR3 sequences; specific embodiments of which inhibit human and/ormurine PCSK9-dependent inhibition of cellular LDL uptake by at least10%. In specific embodiments, the present invention provides homologs ofthe disclosed antagonists characterized as being at least 90% identicalover the heavy and light chain variable region CDR3 sequences to SEQ IDNOs: 17 and 7, respectively; said antagonists which inhibit human and/ormurine PCSK9-dependent inhibition of cellular LDL uptake by at least10%.

Specific embodiments provide isolated PCSK9-specific antagonists and, inmore specific embodiments, antibody molecules which comprise heavy chainvariable region CDR1, CDR2, and CDR3 sequences and light chain variableregion CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 13, 15, 17,3, 5 and 7, respectively; and equivalents thereof characterized ashaving one or more conservative amino acid substitutions in any one ormore of the CDR sequences; specific embodiments of which inhibit humanand/or murine PCSK9-dependent inhibition of cellular LDL uptake by atleast 10%. In specific embodiments, the present invention provideshomologs of the disclosed antagonists characterized as being at least90% identical over the heavy and light chain variable region CDR1, CDR2and CDR3 sequences to SEQ ID NOs: 13, 15, 17, 3, 5 and 7, respectively;said antagonists which inhibit human and/or murine PCSK9-dependentinhibition of cellular LDL uptake by at least 10%.

One particular aspect of the present invention encompasses isolatedPCSK9-specific antagonists and, in more specific embodiments, antibodymolecules which are variants of that disclosed above which comprise aheavy chain variable region CDR3 sequence of SEQ ID NO: 45 wherein theCDR3 sequence is not SEQ ID NO: 17; specific embodiments of whichinhibit human and/or murine PCSK9-dependent inhibition of cellular LDLuptake by at least 10%. Further embodiments hereof additionally compriseheavy chain variable region CDR1 sequence of SEQ ID NO: 43 wherein thevariant sequence is not SEQ ID NO: 13 and/or heavy chain variable regionCDR2 sequence of SEQ ID NO: 44 wherein the variant sequence is not SEQID NO: 15; specific embodiments of which inhibit human and/or murinePCSK9-dependent inhibition of cellular LDL uptake by at least 10%. Inother embodiments, the present invention encompasses heavy chainvariable region sequence comprising CDR1, CDR2, and CDR3 sequence which,respectively, comprises SEQ ID NOs: 43, 44 and 45 in the respectiveregions, which are, respectively, not SEQ ID NOs:13, 15 and 17; specificembodiments of which inhibit human and/or murine PCSK9-dependentinhibition of cellular LDL uptake by at least 10%.

Another aspect of the present invention encompasses isolatedPCSK9-specific antagonists and, in more specific embodiments, antibodymolecules which are variants of that disclosed above which comprise alight chain variable region CDR3 sequence of SEQ ID NO: 48 wherein theCDR3 sequence is not SEQ ID NO: 7; specific embodiments of which inhibithuman and/or murine PCSK9-dependent inhibition of cellular LDL uptake byat least 10%. Further embodiments hereof additionally comprise lightchain variable region CDR1 sequence of SEQ ID NO: 46 wherein the variantsequence is not SEQ ID NO: 3 and/or light chain variable region CDR2sequence of SEQ ID NO: 47 wherein the variant sequence is not SEQ ID NO:5; specific embodiments of which inhibit human and/or murinePCSK9-dependent inhibition of cellular LDL uptake by at least 10%. Inother embodiments, the present invention encompasses light chainvariable region sequence comprising CDR1, CDR2 and CDR3 sequence which,respectively, comprises SEQ ID NOs: 46, 47 and 48 in the respectiveregions, which are, respectively, not SEQ ID NOs: 3, 5 and 7; specificembodiments of which inhibit human and/or murine PCSK9-dependentinhibition of cellular LDL uptake by at least 10%.

Additional distinct embodiments encompass isolated PCSK9-specificantagonists which comprise: (a) a heavy chain variable region comprisingCDR1, CDR2 and CDR3 sequence, wherein (i) the CDR1 sequence comprisesSEQ ID NO: 13 or SEQ ID NO: 43; SEQ ID NO: 43 being different insequence from SEQ ID NO: 13; (ii) the CDR2 sequence comprises SEQ ID NO:15 or SEQ ID NO: 44; SEQ ID NO: 44 being different in sequence from SEQID NO: 15; and (iii) the CDR3 sequence comprises SEQ ID NO: 17 or SEQ IDNO: 45; SEQ ID NO: 45 being different in sequence from SEQ ID NO: 17;and/or (b) a light chain variable region comprising CDR1, CDR2 and CDR3sequence, wherein (i) the CDR1 sequence comprises SEQ ID NO: 3 or SEQ IDNO: 46; SEQ ID NO: 46 being different in sequence from SEQ ID NO: 3;(ii) the CDR2 sequence comprises SEQ ID NO: 5 or SEQ ID NO: 47; SEQ IDNO: 47 being different in sequence from SEQ ID NO: 5; and (iii) the CDR3sequence comprises SEQ ID NO: 7 or SEQ ID NO: 48; SEQ ID NO: 48 beingdifferent in sequence from SEQ ID NO: 7; specific embodiments of whichinhibit human and/or murine PCSK9-dependent inhibition of cellular LDLuptake by at least 10%.

Other aspects of the present invention encompass isolated PCSK9-specificantagonists and, in more specific embodiments, antibody molecules whichare variants of that disclosed above which comprise (i) a heavy chainvariable region sequence comprising CDR1, CDR2, and CDR3 sequence which,respectively, comprises SEQ ID NOs: 43, 44 and 45 in the respectiveregions, which are, respectively, not SEQ ID NOs:13, 15 and 17; and (ii)a light chain variable region sequence comprising CDR1, CDR2 and CDR3sequence which, respectively, comprises SEQ ID NOs: 46, 47 and 48 in therespective regions, which are, respectively, not SEQ ID NOs: 3, 5 and 7;specific embodiments of which inhibit human and/or murinePCSK9-dependent inhibition of cellular LDL uptake by at least 10%.

In specific embodiments herein the CDRs are in place of thecorresponding regions of 1D05 with out without conservative amino acidsubstitutions; specific embodiments of which inhibit human and/or murinePCSK9-dependent inhibition of cellular LDL uptake by at least 10%. Inparticular embodiments, the present invention encompasses isolatedPCSK9-specific antagonists and, in more specific embodiments, antibodymolecules comprising heavy and/or light chain variable regionscomprising SEQ ID NOs: 50 and 49, respectively; said variants SEQ ID NOswhich are not SEQ ID NOs: 11 and 27, respectively; specific embodimentsof which inhibit human and/or murine PCSK9-dependent inhibition ofcellular LDL uptake by at least 10%.

Specific embodiments include any isolated PCSK9-specific antagonist and,in more specific embodiments, antibody molecules which comprise heavychain variable region sequence found in any of SEQ ID NOs: 51-56,optionally comprising a light chain variable region sequence disclosedherein (e.g., SEQ ID NO: 27); specific embodiments of which inhibithuman and/or murine PCSK9-dependent inhibition of cellular LDL uptake byat least 10%. Other embodiments include any isolated PCSK9-specificantagonist and, in more specific embodiments, antibody molecules whichcomprise light chain variable region sequence found in any of SEQ IDNOs: 57-60, optionally comprising a heavy chain variable region sequencedisclosed herein (e.g., SEQ ID NO: 11); specific embodiments of whichinhibit human and/or murine PCSK9-dependent inhibition of cellular LDLuptake by at least 10%.

Particular embodiments are isolated PCSK9-specific antagonists whichcomprise the above-described VH and VL regions in a full lengthantibody. Specific embodiments herein further comprise a series of aminoacids selected from the group consisting of: SEQ ID NO: 21 (IgG1), SEQID NO: 22 (IgG2), SEQ ID NO: 23 (IgG4) and SEQ ID NO: 24 (IgG2m4).

Conservative amino acid substitutions, as one of ordinary skill in theart will appreciate, are substitutions that replace an amino acidresidue with one imparting similar or better (for the intended purpose)functional and/or chemical characteristics. Antagonists bearing suchconservative amino acid substitutions can be tested for retained orbetter activity using functional assays available in the art ordescribed herein. PCSK9-specific antagonists possessing one or moreconservative amino acid substitutions which retain the ability toselectively bind to human PCSK9 and antagonize PCSK9 functioning at alevel the same or better than 1D05 antibody molecules as describedherein are referred to herein as “functional equivalents” of thedisclosed antagonists and form specific embodiments of the presentinvention. Conservative amino acid substitutions are often ones in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Suchmodifications are not designed to significantly reduce or alter thebinding or functional inhibition characteristics of the PCSK9-specificantagonist, albeit they may improve such properties. The purpose formaking a substitution is not significant and can include, but is by nomeans limited to, replacing a residue with one better able to maintainor enhance the structure of the molecule, the charge or hydrophobicityof the molecule, or the size of the molecule. For instance, one maydesire simply to substitute a less desired residue with one of the samepolarity or charge. Such modifications can be introduced by standardtechniques known in the art, such as site-directed mutagenesis andPCR-mediated mutagenesis. One specific means by which those of skill inthe art accomplish conservative amino acid substitutions is alaninescanning mutagenesis as discussed in, for example, MacLennan et al.,1998 Acta Physiol. Scand. Suppl. 643:55-67, and Sasaki et al., 1998 Adv.Biophys. 35:1-24.

In another aspect, the present invention provides isolatedPCSK9-specific antagonists and, in more specific embodiments, antibodymolecules which comprise heavy and/or light chain variable regionscomprising amino acid sequences that are homologous to the correspondingamino acid sequences of the disclosed antibodies, wherein the antibodymolecules inhibit PCSK9-dependent inhibition of cellular LDL uptake.Specific embodiments are antagonists which comprise heavy and/or lightchain variable regions which are at least 90% identical to disclosedheavy and/or light chain variable regions, respectively. Reference to“at least 90% identical” includes at least 90, 91, 92, 93, 94, 95, 96,97, 98, 99 and 100% identical sequences along the full length of themolecule disclosed herein.

PCSK9-specific antagonists with amino acid sequences homologous to theamino acid sequences of antagonists described herein are typicallyproduced to improve one or more of the properties of the antagonistwithout negatively impacting its specificity for PCSK9. One method ofobtaining such sequences, which is not the only method available to theskilled artisan, is to mutate sequence encoding the PCSK9-specificantagonist or specificity-determining region(s) thereof, express anantagonist comprising the mutated sequence(s), and test the encodedantagonist for retained function using available functional assaysincluding those described herein. Mutation may be by site-directed orrandom mutagenesis. As one of skill in the art will appreciate, however,other methods of mutagenesis can readily bring about the same effect.For example, in certain methods, the spectrum of mutants are constrainedby non-randomly targeting conservative substitutions based on eitheramino acid chemical or structural characteristics, or else by proteinstructural considerations. In affinity maturation experiments, severalsuch mutations may be found in a single selected molecule, whether theyare randomly or non-randomly selected. There are also variousstructure-based approaches toward affinity maturation as demonstratedin, e.g., U.S. Pat. No. 7,117,096, PCT Pub. Nos.: WO 02/084277 and WO03/099999; the disclosures of which are incorporated herein byreference.

As used herein, the percent homology between two amino acid or nucleicacid sequences is equivalent to the percent identity between the twosequences, and these two terms will be used interchangeably throughout.As used herein, % identity of two nucleic acid or amino acid sequencesis determined using the algorithm of Karlin and Altschul (Proc. Natl.Acad. Sci. USA 90:5873-5877, 1993). Such an algorithm is incorporatedinto the NBLAST and XBLAST programs of Altschul et al., 1990 J. Mol.Biol. 215:403-410. BLAST nucleotide searches are performed with theNBLAST program, score=100, wordlength=12, to obtain nucleic acidsequences homologous to a nucleic acid molecule of the invention. BLASTprotein searches are performed with the XBLAST program, score=50,wordlength=3, to obtain amino acid sequences homologous to an amino acidsequence disclosed herein. To obtain gapped alignments for comparisonpurposes, Gapped BLAST is utilized as described in Altschul et al., 1997Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) are used.

Utilization of components of one or more disclosed PCSK9-specificmolecules to produce other binding molecules with similar or betterspecificity is well within the realm of one skilled in the art. This canbe accomplished, for example, using techniques of recombinant DNAtechnology. One specific example of this involves the introduction ofDNA encoding the immunoglobulin variable region, or one or more of theCDRs, of an antibody to the variable region, constant region, orconstant region plus framework regions, as appropriate, of a differentimmunoglobulin. Such molecules form important aspects of the presentinvention. Specific immunoglobulins or the corresponding sequences, intowhich particular disclosed sequences may be inserted or, in thealternative, form the essential part of, include but are not limited tothe following antibody molecules which form particular embodiments ofthe present invention: a Fab (monovalent fragment with variable light(VL), variable heavy (VH), constant light (CL) and constant heavy 1(CH1) domains), a F(ab′)₂ (bivalent fragment comprising two Fabfragments linked by a disulfide bridge or alternative at the hingeregion), a Fd (VH and CH1 domains), a Fv (VL and VH domains), a scFv (asingle chain Fv where VL and VH are joined by a linker, e.g., a peptidelinker, see, e.g., Bird et al., 1988 Science 242:423-426, Huston et al.,1988 PNAS USA 85:5879-5883), a bispecific antibody molecule (an antibodymolecule comprising a PCSK9-specific antibody or antigen bindingfragment as disclosed herein linked to a second functional moiety havinga different binding specificity than the antibody, including, withoutlimitation, another peptide or protein such as an antibody, or receptorligand), a bispecific single chain Fv dimer (see, e.g., PCT/US92/09965),an isolated CDR3, a minibody (single chain-CH3 fusion that selfassembles into a bivalent dimer of about 80 kDa), a ‘scAb’ (an antibodyfragment containing VH and VL as well as either CL or CH1), a dAbfragment (VH domain, see, e.g., Ward et al., 1989 Nature 341:544-546,and McCafferty et al., 1990 Nature 348:552-554; or VL domain; Holt etal., 2003 Trends in Biotechnology 21:484-489), a diabody (see, e.g.,Holliger et al., 1993 PNAS USA 90:6444-6448 and InternationalApplication Number WO 94/13804), a triabody, a tetrabody, a minibody (ascFv joined to a CH3; see, e.g., Hu et al., 1996 Cancer Res.56:3055-3061), IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgD, IgA, IgE or anyderivatives thereof, and artificial antibodies based upon proteinscaffolds, including but not limited to fibronectin type III polypeptideantibodies (see, e.g., U.S. Pat. No. 6,703,199 and InternationalApplication Number WO 02/32925) or cytochrome B; see, e.g., Koide etal., 1998 J. Molec. Biol. 284:1141-1151, and Nygren et al., 1997 CurrentOpinion in Structural Biology 7:463-469; the disclosures of which areincorporated herein by reference. Certain antibody molecules including,but not limited to, Fv, scFv, diabody molecules or domain antibodies(Domantis) may be stabilized by incorporating disulfide bridges to linethe VH and VL domains, see, e.g., Reiter et al., 1996 Nature Biotech.14:1239-1245; the disclosure of which is incorporated herein byreference. Bispecific antibodies may be produced using conventionaltechnologies (see, e.g., Holliger & Winter, 1993 Current OpinionBiotechnol. 4:446-449, specific methods of which include productionchemically, or from hybrid hybridomas) and other technologies including,but not limited to, the BiTE™ technology (molecules possessing antigenbinding regions of different specificity with a peptide linker) andknobs-into-holes engineering (see, e.g., Ridgeway et al., 1996 ProteinEng. 9:616-621; the disclosure of which is incorporated herein byreference). Bispecific diabodies may be produced in E. coli, and thesemolecules as other PCSK9-specific antagonists, as one of skill in theart will appreciate, may be selected using phage display in theappropriate libraries (see, e.g., International Application Number WO94/13804; the disclosure of which is incorporated herein by reference).

Variable domains, into which CDRs of interest are inserted, may beobtained from any germ-line or rearranged human variable domain.Variable domains may also be synthetically produced. The CDR regions canbe introduced into the respective variable domains using recombinant DNAtechnology. One means by which this can be achieved is described inMarks et al., 1992 Bio/Technology 10:779-783; the disclosure of which isincorporated herein by reference. A variable heavy domain may be pairedwith a variable light domain to provide an antigen binding site. Inaddition, independent regions (e.g., a variable heavy domain alone) maybe used to bind antigen. The artisan is well aware, as well, that twodomains of an Fv fragment, VL and VH, while perhaps coded by separategenes, may be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent molecules (scFvs).

Specific embodiments provide the CDR(s) in germline framework regions.Framework regions, including but not limited to human framework regions,are known to those of skill in the art (e.g., a human or non-humanframework). The framework regions may be naturally occurring orconsensus framework regions. In one aspect, the framework region of anantibody of the invention is human (see, e.g., Clothia et al., 1998 J.Mol. Biol. 278:457-479 for a listing of human framework regions; saiddisclosure of which is incorporated herein by reference in itsentirety). Specific embodiments herein provide heavy chain variable CDR3SEQ ID NO: 17 into VH1A_(—)3 in place of the relevant CDR. Specificembodiments herein provide heavy chain variable CDR1, CDR2 and/or CDR3sequences (SEQ ID NO:s 13, 15 and 17, respectively) into VH1A_(—)3 inplace of the relevant CDRs. Specific embodiments herein provide lightchain variable CDR3 SEQ ID NO: 7 into VK1_(—)4 in place of the relevantCDR. Specific embodiments herein provide light chain variable CDR1, CDR2and/or CDR3 sequences (SEQ ID NO:s 3, 5 and 7, respectively) intoVK1_(—)4 in place of the relevant CDRs. Specific embodiments furtherprovide heavy chain variable CDR3 SEQ ID NO: 17 and light chain variableCDR3 SEQ ID NO: 7 into VH1A_(—)3 and VK1_(—)4 germline sequences,respectively. Further embodiments, provide heavy chain variable CDR1,CDR2 and/or CDR3 sequences (SEQ ID NO:s 13, 15 and 17, respectively)into VH1A_(—)3 in place of the relevant CDRs; and light chain variableCDR1, CDR2 and/or CDR3 sequences (SEQ ID NO:s 3, 5 and 7, respectively)into VK1_(—)4 in place of the relevant CDRs.

The present invention encompasses antibody molecules that are human,humanized, deimmunized, chimeric and primatized. The invention alsoencompasses antibody molecules produced by the process of veneering;see, e.g., Mark et al., 1994 Handbook of Experimental Pharmacology, vol.113: The pharmacology of monoclonal Antibodies, Springer-Verlag, pp.105-134; the disclosure of which is incorporated herein by reference.“Human” in reference to the disclosed antibody molecules specificallyrefers to antibody molecules having variable and/or constant regionsderived from human germline immunoglobulin sequences, wherein saidsequences may, but need not, be modified/altered to have certain aminoacid substitutions or residues that are not encoded by human germlineimmunoglobulin sequence. Such mutations can be introduced by methodsincluding, but not limited to, random or site-specific mutagenesis invitro, or by somatic mutation in vivo. Specific examples of mutationtechniques discussed in the literature are that disclosed in Gram etal., 1992 PNAS USA 89:3576-3580; Barbas et al., 1994 PNAS USA91:3809-3813, and Schier et al., 1996 J. Mol. Biol. 263:551-567; thedisclosures of which are incorporated herein by reference. These areonly specific examples and do not represent the only availabletechniques. There are a plethora of mutation techniques in thescientific literature which are available to, and widely appreciated by,the skilled artisan. “Humanized” in reference to the disclosed antibodymolecules refers specifically to antibody molecules wherein CDRsequences derived from another mammalian species, such as a mouse, aregrafted onto human framework sequences. “Primatized” in reference to thedisclosed antibody molecules refers to antibody molecules wherein CDRsequences of a non-primate are inserted into primate frameworksequences, see, e.g., WO 93/02108 and WO 99/55369; the disclosures ofwhich are incorporated herein by reference.

Specific antibodies of the present invention are monoclonal antibodiesand, in particular embodiments, are in one of the following antibodyformats: IgD, IgA, IgE, IgM, IgG1, IgG2, IgG3, IgG4 or any derivative ofany of the foregoing. The language “derivatives thereof” or“derivatives” in this respect includes, inter alia, (i) antibodies andantibody molecules with conservative modifications in one or bothvariable regions (i.e., VH and/or VL), (ii) antibodies and antibodymolecules with manipulations in the constant regions of the heavy and/orlight chains, and/or (iii) antibodies and antibody molecules thatcontain additional chemical moieties which are not normally a part ofthe immunoglobulin molecule (e.g., pegylation).

Manipulations of the variable regions can be within one or more of theVH and/or VL CDR regions. Site-directed mutagenesis, random mutagenesisor other method for generating sequence or molecule diversity can beutilized to create mutants which can subsequently be tested for aparticular functional property of interest in available in vitro or invivo assays including those described herein.

Antibodies of the present invention also include those in whichmodifications have been made to the framework residues within VH and/orVL to improve one or more properties of the antibody of interest.Typically, such framework modifications are made to decrease theimmunogenicity of the antibody. For example, one approach is to“backmutate” one or more framework residues to the correspondinggermline sequence. More specifically, an antibody that has undergonesomatic mutation may contain framework residues that differ from thegermline sequence from which the antibody is derived. Such residues canbe identified by comparing the antibody framework sequences to thegermline sequences from which the antibody is derived. Such“backmutated” antibodies are also intended to be encompassed by theinvention. Another type of framework modification involves mutating oneor more residues within the framework region, or even within one or moreCDR regions, to remove T cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Carr et al; the disclosure of which isincorporated herein by reference.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention may be engineered to includemodifications within the Fe or constant regions, where present,typically to alter one or more functional properties of the antibody,such as serum half-life, complement fixation, Fc receptor binding,and/or antigen-dependent cellular cytotoxicity.

The concept of generating “hybrids” or “combinatorial” IgG formscomprising various antibody isotypes to hone in on desired effectorfunctionality has generally been described; see, e.g., Tao et al., 1991J. Exp. Med. 173:1025-1028. A specific embodiment of the presentinvention encompasses antibody molecules that possess specificmanipulations in the Fc region which have been found to result inreduced or altered binding to FcγR receptors, C1q or FcRn on the part ofthe antibody. The present invention, therefore, encompasses antibodiesin accordance with the present description that do not provoke (orprovoke to a lesser extent) antibody-dependent cellular cytotoxicity(“ADCC”), complement-mediated cytotoxicity (“CMC”), or form immunecomplexes, while retaining normal pharmacokinetic (“PK”) properties.Specific embodiments of the present invention provide an antibodymolecule as defined in accordance with the present invention whichcomprises, as part of its immunoglobulin structure, SEQ ID NO: 24 and,in particular embodiments, residues 107-326 of SEQ ID NO: 24 as part ofthe immunoglobulin structure. The present invention encompasses antibodymolecules which comprise: (i) a light chain comprising SEQ ID NO: 1, and(ii) a heavy chain comprising SEQ ID NO: 11 in sequence with (adjacentto) or followed by a series of amino acids selected from the groupconsisting of: SEQ ID NO: 21 (IgG1), SEQ ID NO: 22 (IgG2), SEQ ID NO: 23(IgG4) and SEQ ID NO: 24 (IgG2m4). FIG. 6 illustrates a comparison ofsequence comprising SEQ ID NO: 24, particularly IgG2m4, with IgG1, IgG2,and IgG4. Amino acid sequences for mature, secreted anti-PCSK9 IgG2m4heavy and light chains can be found as SEQ ID NOs: 25 and 26,respectively. Antibody molecules encoded at least in part by saidsequence are encompassed herein.

Specific PCSK9-specific antagonists may carry a detectable label, or maybe conjugated to a toxin (e.g., a cytotoxin), a radioactive isotope, aradionuclide, a liposome, a targeting moiety, a biosensor, a cationictail, or an enzyme (e.g., via a peptidyl bond or linker). SuchPCSK9-specific antagonist compositions form an additional aspect of thepresent invention.

In another aspect, the present invention provides isolated nucleic acidencoding disclosed PCSK9-specific antagonists. “Isolated” as mentionedprior refers to the property of the thing referred to that makes themdifferent from that found in nature. The difference can be, for example,that they are of a different purity than that found in nature, or thatthey are of a different structure or form part of a different structurethan that found in nature. An example of nucleic acid not found innature is, for example, nucleic acid substantially free of othercellular material. The nucleic acid may be present in whole cells, in acell lysate, or in a partially purified or substantially pure form. Inspecific instances, a nucleic acid may be isolated when purified awayfrom other cellular components or other contaminants, e.g., othercellular nucleic acids or proteins, for example, using standardtechniques, including without limitation, alkaline/SDS treatment, CsC1banding, column chromatography, agarose gel electrophoresis and othersuitable methods known in the art. The nucleic acid may include DNA(inclusive of cDNA) and/or RNA. Nucleic acids of the present inventioncan be obtained using standard molecular biology techniques. Forantibodies expressed by hybridomas (e.g., hybridomas prepared fromtransgenic mice carrying human immunoglobulin genes), cDNAs encoding thelight and heavy chains of the antibody made by the hybridoma can beobtained by standard PCR amplification or cDNA cloning techniques. Forantibodies obtained from an immunoglobulin gene library (e.g., usingphage display techniques), nucleic acid encoding the antibody can berecovered from the library.

The present invention encompasses isolated nucleic acid encodingdisclosed variable heavy and/or light chains and select componentsthereof, particularly the disclosed variable or respective CDR regionsand, in particular CDR3. In specific embodiments hereof, the CDR(s) areprovided within antibody framework regions and, in particularembodiments, human framework regions. Specific embodiments provideisolated nucleic acid encoding the CDR(s) into germline frameworkregions including, but not limited to, human germline framework regions.Specific embodiments herein provide isolated nucleic acid encoding heavychain CDR SEQ ID NO: 17 (in specific embodiments, said nucleic acid ofwhich comprises SEQ ID NO: 18) into VH1A_(—)3 in place of the nucleicacid encoding the relevant CDR. Specific embodiments herein providenucleic acid encoding heavy chain variable CDR1, CDR2 and/or CDR3sequences SEQ ID NOs: 13, 15 and 17, respectively (and, in particularembodiments, said nucleic acid of which comprises SEQ ID NOs: 14, 16 and18, respectively) into VH1A_(—)3 in place of the relevant CDRs. Specificembodiments herein provide isolated nucleic encoding light chain CDR SEQID NO: 7 (in specific embodiments, said nucleic acid of which comprisesSEQ ID NO: 8) into VK1_(—)4 in place of the nucleic acid encoding therelevant CDR. Specific embodiments herein provide nucleic acid encodinglight chain variable CDR1, CDR2 and/or CDR3 sequences SEQ ID NOs: 3, 5and 7, respectively (and, in particular embodiments, said nucleic acidof which comprises SEQ ID NOs: 4, 6 and 8, respectively) into VK1_(—)4in place of the relevant CDRs. Specific embodiments further provideheavy chain variable CDR3 SEQ ID NO: 17 (and, in particular embodiments,said nucleic acid of which comprises SEQ ID NO: 18) and light chainvariable CDR3 SEQ ID NO: 7 (and, in particular embodiments, said nucleicacid of which comprises SEQ ID NO: 8) into VH1A_(—)3 and VK1_(—)4germline sequences, respectively. Further embodiments provide heavychain variable CDR1, CDR2 and/or CDR3 sequences SEQ ID NOs: 13, 15 and17, respectively (and, in particular embodiments, said nucleic acid ofwhich comprises SEQ ID NOs: 14, 16 and 18, respectively) into VH1A_(—)3in place of the relevant CDRs; and light chain variable CDR1, CDR2and/or CDR3 sequences SEQ ID NOs: 3, 5 and 7, respectively (and, inparticular embodiments, said nucleic acid of which comprises SEQ ID NOs:4, 6 and 8, respectively) into VK1_(—)4 in place of the relevant CDRs.

The isolated nucleic acid encoding the variable regions can be providedwithin any desired antibody molecule format including, but not limitedto, the following: F(ab′)₂, a Fab, a Fv, a scFv, bispecific antibodymolecules (antibody molecules comprising a PCSK9-specific antibody orantigen binding fragment as disclosed herein linked to a secondfunctional moiety having a different binding specificity than theantibody, including, without limitation, another peptide or protein suchas an antibody, or receptor ligand), a bispecific single chain Fv dimer,a minibody, a dAb fragment, diabody, triabody or tetrabody, a minibody,IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgD, IgA, IgE or any derivativesthereof.

Specific embodiments provide isolated nucleic acid which encodesPCSK9-specific antagonists and, in more specific embodiments, antibodymolecules comprising a heavy chain variable domain which comprises SEQID NO: 11; specific embodiments of which comprise nucleic acid sequenceSEQ ID NO: 12. Specific embodiments of the present invention provideisolated nucleic acid encoding PCSK9-specific antagonists and, in morespecific embodiments, antibody molecules, which additionally comprise:(i) nucleic acid encoding heavy chain CDR1 amino acid sequence SEQ IDNO: 13 (specific embodiments of which comprise nucleic acid SEQ ID NO:14) and/or (ii) nucleic acid encoding heavy chain CDR2 amino acidsequence SEQ ID NO: 15 (specific embodiments of which comprise nucleicacid SEQ ID NO: 16). Specific embodiments provide isolated nucleic acidencoding PCSK9-specific antagonists and, in more specific embodiments,antibody molecules comprising a light chain variable domain whichcomprises SEQ ID NO: 27; specific embodiments of which comprise nucleicacid sequence SEQ ID NO: 28. Specific embodiments of the presentinvention provide isolated nucleic acid encoding PCSK9-specificantagonists and, in more specific embodiments, antibody molecules, whichadditionally comprise: (i) nucleic acid encoding light chain CDR1 aminoacid sequence SEQ ID NO: 3 (specific embodiments of which comprisenucleic acid SEQ ID NO: 4) and/or (ii) nucleic acid encoding light chainCDR2 amino acid sequence SEQ ID NO: 5 (specific embodiments of whichcomprise nucleic acid SEQ ID NO: 6). Specific embodiments provideisolated nucleic acid encoding PCSK9-specific antagonists and, in morespecific embodiments, antibody molecules which comprise a heavy chainvariable domain which comprises SEQ ID NO: 11; specific embodiments ofwhich comprise nucleic acid sequence SEQ ID NO: 12; and a light chainvariable domain which comprises SEQ ID NO: 27; specific embodiments ofwhich comprise nucleic acid sequence SEQ ID NO: 28. Specific embodimentsprovide isolated nucleic acid encoding (i) heavy chain CDR1, CDR2 and/orCDR3 sequences (SEQ ID NOs: 13, 15 and 17, respectively; specificembodiments of which comprise nucleic acid SEQ ID NOs: 14, 16 and/or 18,respectively) preferably in a framework region (including but notlimited to a human framework region); and (ii) light chain CDR1, CDR2and/or CDR3 sequences (SEQ ID NO: 3, 5 and 7, respectively; specificembodiments of which comprise nucleic acid SEQ ID NOs: 4, 6 and/or 8,respectively) preferably in a framework region (including but notlimited to a human framework region). The present invention furtherprovides in specific embodiments, homologs of the antagonists disclosedabove, characterized as being at least 90% identical over the heavyand/or light chain variable regions, or the CDR regions, as appropriate,whichever is present to the corresponding sequences of 1D05.

Additional embodiments provide isolated nucleic acid encodingPCSK9-specific antagonists and, in more specific embodiments, antibodymolecules which comprise a light chain comprising SEQ ID NO: 1 (specificembodiments of which comprise nucleic acid SEQ ID NO: 2) and a heavychain or Fd chain comprising amino acids 1-233 of SEQ ID NO: 9, or SEQID NO: 9 (specific embodiments of which comprise nucleic acid 1-699 ofSEQ ID NO: 10, or SEQ ID NO: 10, respectively). Further embodimentsprovide isolated nucleic acid encoding PCSK9-specific antagonists and,in more specific embodiments, antibody molecules which comprise a lightchain comprising SEQ ID NO: 26 (specific embodiments of which compriseSEQ ID NO: 30) and a heavy chain comprising SEQ ID NO: 25 (specificembodiments of which comprise SEQ ID NO: 29). The present inventionfurther provides in specific embodiments, homologs of the antagonistsdisclosed above, characterized as being at least 90% identical over theheavy and/or light chains to the corresponding sequences of 1D05.

Specific embodiments of the present invention encompass nucleic acidencoding antibody molecules that possess manipulations in the Fc regionwhich result in reduced or altered binding to FcγR receptors, C1q, orFcRn on the part of the antibody. One specific embodiment of the presentinvention is isolated nucleic acid which encodes for antibody moleculescomprising as part of their immunoglobulin structure SEQ ID NO: 24 and,in particular embodiments, residues 107-326 of SEQ ID NO: 24. Inspecific embodiments, synthetic PCSK9-specific antagonists can beproduced by expression from nucleic acid generated from oligonucleotidessynthesized and assembled within suitable expression vectors; see, e.g.,Knappick et al., 2000 J. Mol. Biol. 296:57-86, and Krebs et al., 2001 J.Immunol. Methods 254:67-84.

The present invention encompasses nucleic acid encoding antibodymolecules which comprise: (i) nucleic acid encoding a light chaincomprising SEQ ID NO: 1 (specific embodiments of which comprise nucleicacid SEQ ID NO: 2), and (ii) nucleic acid encoding a heavy chaincomprising SEQ ID NO: 11 (specific embodiments of which comprise nucleicacid SEQ ID NO: 12) followed in sequence by (adjacent to) a set ofnucleotides encoding for a set of amino acids selected from the groupconsisting of: SEQ ID NO: 21 (IgG1), SEQ ID NO: 22 (IgG2), SEQ ID NO: 23(IgG4) and SEQ ID NO: 24 (IgG2m4). Nucleotide sequences for mature,secreted anti-PCSK9 IgG2m4 heavy and light chains can be found as SEQ IDNOs: 29 and 30, respectively. Plasmid sequences comprising heavy andlight chain 1D05 anti-PCSK9 IgG2m4 antibody molecules can be found asSEQ ID NOs: 31 and 32, respectively. Nucleic acid encoding such antibodymolecules form important embodiments hereof.

Also included within the present invention are isolated nucleic acidscomprising nucleotide sequences which are at least about 90% identicaland more preferably at least about 95% identical to the full length ofthe nucleotide sequences described herein, and which nucleotidesequences encode PCSK9-specific antagonists which inhibitPCSK9-dependent inhibition of cellular LDL uptake by at least 10%.

Reference to “at least about 90% identical” throughout the applicationincludes at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%identical.

The invention further provides isolated nucleic acid at least a portionof which hybridizes to the complement of nucleic acid consisting of SEQID NO: 12 and/or SEQ ID NO: 28 under stringent hybridization conditions,said nucleic acid of which confers upon antibody molecules the abilityto specifically bind PCSK9 and antagonize PCSK9 function, andPCSK9-specific antagonists expressed employing said nucleic acid.Methods for hybridizing nucleic acids are well-known in the art; see,e.g., Ausubel, Current Protocols in Molecular Biology, John Wiley &Sons, N.Y., 6.3.1-6.3.6, 1989. Stringent hybridization conditionsinvolve hybridizing at 68° C. in 5×SSC/5×Denhardt's solution (orequivalent)/1.0% SDS, and washing in 0.2× SSC/0.1% SDS at roomtemperature. Moderately stringent conditions include washing in 3×SSC at42° C. The parameters of salt concentration and temperature can bevaried to achieve the optimal level of identity between the probe andthe target nucleic acid. The skilled artisan can manipulate varioushybridization and/or washing conditions to specifically target nucleicacid in the hybridizing portion that is at least 80, 85, 90, 95, 98, or99% identical to SEQ ID NO: 12 and/or SEQ ID NO: 28. Basic parametersaffecting the choice of hybridization conditions and guidance fordevising suitable conditions are set forth by Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., chapters 9 and 11, 1989 and Ausubel et al. (eds),Current Protocols in Molecular Biology, John Wiley & Sons, Inc.,sections 2.10 and 6.3-6.4, 1995 (the disclosures of which areincorporated herein by reference), and can be readily determined bythose having ordinary skill in the art. PCSK9 antagonists having one ormore variable regions comprising nucleic acid which hybridizes to thecomplement of nucleic acid consisting of SEQ ID NO: 12 and/or SEQ ID NO:28 under stringent hybridization conditions should be effective inantagonizing one or more functions of PCSK9. Said antagonists andencoding nucleic acid, thus, form important embodiments of the presentinvention.

In another aspect, the present invention provides vectors comprising thenucleic acid disclosed herein. Vectors in accordance with the presentinvention include, but are not limited to, plasmids and other expressionconstructs (e.g., phage or phagemid, as appropriate) suitable for theexpression of the desired antibody molecule at the appropriate level forthe intended purpose; see, e.g., Sambrook & Russell, Molecular Cloning:A Laboratory Manual: 3^(rd) Edition, Cold Spring Harbor LaboratoryPress; the disclosure of which is incorporated herein by reference. Formost cloning purposes, DNA vectors may be used. Typical vectors includeplasmids, modified viruses, bacteriophage, cosmids, yeast artificialchromosomes, bacterial artificial chromosomes, and other forms ofepisomal or integrated DNA. It is well within the purview of the skilledartisan to determine an appropriate vector for a particular genetransfer, generation of a recombinant PCSK9-specific antagonist, orother use. In specific embodiments, in addition to a recombinant gene,the vector may also contain an origin of replication for autonomousreplication in a host cell, appropriate regulatory sequences, such as apromoter, a termination sequence, a polyadenylation sequence, anenhancer sequence, a selectable marker, a limited number of usefulrestriction enzyme sites, and/or other sequences as appropriate and thepotential for high copy number. Examples of expression vectors for theproduction of protein-specific antagonists are well known in the art;see, e.g., Persic et al., 1997 Gene 187:9-18; Boel et al., 2000 J.Immunol. Methods 239:153-166, and Liang et al., 2001 J. Immunol. Methods247:119-130; the disclosures of which are incorporated herein byreference. If desired, nucleic acid encoding the antagonist may beintegrated into the host chromosome using techniques well known in theart; see, e.g., Ausubel, Current Protocols in Molecular Biology, JohnWiley & Sons, 1999, and Marks et al., International Application NumberWO 95/17516. Nucleic acid may also be expressed on plasmids maintainedepisomally or incorporated into an artificial chromosome; see, e.g.,Csonka et al., 2000 J. Cell Science 113:3207-3216; Vanderbyl et al.,2002 Molecular Therapy 5:10. Specifically with regards to antibodymolecules, the antibody light chain gene and the antibody heavy chaingene can be inserted into separate vectors or, more typically, bothgenes may be inserted into the same expression vector. Nucleic acidencoding any PCSK9-specific antagonist or component thereof can beinserted into an expression vector using standard methods (e.g.,ligation of complementary restriction sites on the nucleic acid fragmentand vector, or blunt end ligation if no restriction sites are present).Another specific example of how this may be carried out is through useof recombinational methods, e.g. the Clontech “InFusion” system, orInvitrogen “TOPO” system (both in vitro), or intracellularly (e.g. theCre-Lox system). Specifically with regards to antibody molecules, thelight and heavy chain variable regions can be used to create full-lengthantibody genes of any antibody isotype by inserting them into expressionvectors already encoding heavy chain constant and light chain constantregions of the desired isotype such that the VH segment is operativelylinked to the CH segment(s) within the vector and the VL segment isoperatively linked to the CL segment within the vector. Additionally oralternatively, the recombinant expression vector comprising nucleic acidencoding a PCSK9-specific antagonist can encode a signal peptide thatfacilitates secretion of the antagonist from a host cell. The nucleicacid can be cloned into the vector such that the nucleic acid encoding asignal peptide is linked in-frame adjacent to the PCSK9-specificantagonist-encoding nucleic acid. The signal peptide may be animmunoglobulin or a non-immunoglobulin signal peptide. Any techniqueavailable to the skilled artisan may be employed to introduce thenucleic acid into the host cell; see, e.g., Morrison, 1985 Science,229:1202. Methods of subcloning nucleic acid molecules of interest intoexpression vectors, transforming or transfecting host cells containingthe vectors, and methods of making substantially pure protein comprisingthe steps of introducing the respective expression vector into a hostcell, and cultivating the host cell under appropriate conditions arewell known. The PCSK9-specific antagonist so produced may be harvestedfrom the host cells in conventional ways. Techniques suitable for theintroduction of nucleic acid into cells of interest will depend on thetype of cell being used. General techniques include, but are not limitedto, calcium phosphate transfection, DEAE-Dextran, electroporation,liposome-mediated transfection and transduction using virusesappropriate to the cell line of interest (e.g., retrovirus, vaccinia,baculovirus, or bacteriophage).

In another aspect, the present invention provides isolated cell(s)comprising nucleic acid encoding disclosed PCSK9-specific antagonists. Avariety of different cell lines are contemplated herein and can be usedfor the recombinant production of PCSK9-specific antagonists, includingbut not limited to those from prokaryotic organisms (e.g., E. coli,Bacillus, and Streptomyces) and from eukaryotic (e.g., yeast,Baculovirus, and mammalian); see, e.g., Breitling et al., Recombinantantibodies, John Wiley & Sons, Inc. and Spektrum Akademischer Verlag,1999; the disclosure of which is incorporated herein by reference. Plantcells, including transgenic plants, and animal cells, includingtransgenic animals (other than humans), comprising the nucleic acid orantagonists disclosed herein are also contemplated as part of thepresent invention. Suitable mammalian cells or cell lines including, butnot limited to, those derived from Chinese Hamster Ovary (CHO cells,including but not limited to DHFR-CHO cells (described in Urlaub andChasin, 1980 Proc. Natl. Acad. Sci. USA 77:4216-4220) used, for example,with a DHFR selectable marker (e.g., as described in Kaufman and Sharp,1982 Mol. Biol. 159:601-621), NS0 myeloma cells (where a GS expressionsystem as described in WO 87/04462, WO 89/01036, and EP 338,841 may beused), COS cells, SP2 cells, HeLa cells, baby hamster kidney cells,YB2/0 rat myeloma cells, human embryonic kidney cells, human embryonicretina cells, and others comprising the nucleic acid or antagonistsdisclosed herein form additional embodiments of the present invention;the preceding cited disclosures of which are incorporated herein byreference. Specific embodiments of the present invention comprisingnucleic acid encoding disclosed PCSK9-specific antagonists include, butare not limited to, E. coli; see, e.g., Pliickthun, 1991 Bio/Technology9:545-551, or yeast, such as Pichia, and recombinant derivatives thereof(see, e.g., Li et al., 2006 Nat. Biotechnol. 24:210-215); the precedingdisclosures of which are incorporated herein by reference. Specificembodiments of the present invention relate to eukaryotic cellscomprising nucleic acid encoding the disclosed PCSK9-specificantagonists, see, Chadd & Chamow, 2001 Current Opinion in Biotechnology12:188-194, Andersen & Krummen, 2002 Current Opinion in Biotechnology13:117, Larrick & Thomas, 2001 Current Opinion in Biotechnology12:411-418; the disclosures of which are incorporated herein byreference. Specific embodiments of the present invention relate tomammalian cells comprising nucleic acid encoding the disclosedPCSK9-specific antagonists which are able to produce PCSK9-specificantagonists with proper post translational modifications. Posttranslational modifications include, but are by no means limited to,disulfide bond formation and glycosylation. Another type of posttranslational modification is signal peptide cleavage. Preferredembodiments herein have the appropriate glycosylation; see, e., Yoo etal., 2002 J. Immunol. Methods 261:1-20; the disclosure of which isincorporated herein by reference. Naturally occurring antibodies containat least one N-linked carbohydrate attached to a heavy chain. Id.Different types of mammalian host cells can be used to provide forefficient post-translational modifications. Examples of such host cellsinclude Chinese Hamster Ovary (CHO), HeLa, C6, PC12, and myeloma cells;see, Yoo et al., 2002 J. Immunol. Methods 261:1-20, and Persic et al.,1997 Gene 187:9-18; the disclosures of which are incorporated herein byreference.

In another aspect, the present invention provides isolated cell(s)comprising a polypeptide of the present invention.

In another aspect, the present invention provides a method of making aPCSK9-specific antagonist of the present invention, which comprisesincubating a cell comprising nucleic acid encoding the PCSK9-specificantagonist, or a heavy and/or light chain or a fragment thereof (e.g.,VH and/or VL, or one or more of the disclosed heavy and/or light chainvariable region CDRs) of a desired PCSK9-specific antagonist (dictatedby the desired antagonist) with specificity for human and/or murinePCSK9 under conditions that allow the expression of the PCSK9-specificantagonist, or the expression and assembly of said heavy and/or lightchains or fragment into a PCSK9-specific antagonist, and isolating saidPCSK9-specific antagonist from the cell. One example by which togenerate particular desired heavy and/or light chain sequence orfragment is to first amplify (and modify) the germline heavy and/orlight chain variable sequences or fragment using PCR. Germline sequencefor human heavy and/or light variable regions are readily available tothe skilled artisan, see, e.g., the “Vbase” human germline sequencedatabase, and Kabat, E. A. et al., 1991 Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242; Tomlinson, I. M. et al.,1992 “The Repertoire of Human Germline VH Sequences Reveals about FiftyGroups of VH Segments with Different Hypervariable Loops” J. Mol. Biol.227:776-798; and Cox, J. P. L. et al., 1994 “A Directory of HumanGerm-line V_(κ) Segments Reveals a Strong Bias in their Usage” Eur. J.Immunol. 24:827-836; the disclosures of which are incorporated herein byreference. Mutagenesis of germline sequences may be carried out usingstandard methods, e.g., PCR-mediated mutagenesis where the mutations areincorporated into PCR primers, or site-directed mutagenesis. Iffull-length antibodies are desired, sequence is available for the humanheavy chain constant region genes; see, e.g., Kabat. E. A. et al., 1991Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242.Fragments containing these regions may be obtained, for example, bystandard PCR amplification. Alternatively, the skilled artisan can availhim/herself of vectors already encoding heavy and/or light chainconstant regions.

Available techniques exist to recombinantly produce other antibodymolecules which retain the specificity of an original antibody. Aspecific example of this is where DNA encoding the immunoglobulinvariable region or the CDRs is introduced into the constant regions, orconstant regions and framework regions, or simply the framework regions,of another antibody molecule; see, e.g., EP-184,187, GB 2188638, andEP-239400; the disclosures of which are incorporated herein byreference. Cloning and expression of antibody molecules, includingchimeric antibodies, are described in the literature; see, e.g., EP0120694 and EP 0125023; the disclosures of which are incorporated hereinby reference.

Antibody molecules in accordance with the present invention may, in oneinstance, be raised and then screened for characteristics identifiedherein using known techniques. Basic techniques for the preparation ofmonoclonal antibodies are described in the literature, see, e.g., Kohlerand Milstein (1975, Nature 256:495-497); the disclosure of which isincorporated herein by reference. Fully human monoclonal antibodies canbe produced by available methods. These methods include, but are by nomeans limited to, the use of genetically engineered mouse strains whichpossess an immune system whereby the mouse antibody genes have beeninactivated and in turn replaced with a repertoire of functional humanantibody genes, while leaving other components of the mouse immunesystem unchanged. Such genetically engineered mice allow for the naturalin vivo immune response and affinity maturation process which results inhigh affinity, full human monoclonal antibodies. This technology is wellknown in the art and is fully detailed in various publications,including but not limited to U.S. Pat. Nos. 5,545,806; 5,569,825;5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318;5,874,299; 5,770,249 (assigned to GenPharm International and availablethrough Medarex, under the umbrella of the “UltraMab Human AntibodyDevelopment System”); as well as U.S. Pat. Nos. 5,939,598; 6,075,181;6,114,598; 6,150,584 and related family members (assigned to Abgenix,disclosing their XenoMouse® technology); the disclosures of which areincorporated herein by reference. See also reviews from Kellerman andGreen, 2002 Curr. Opinion in Biotechnology 13:593-597, and Kontermann &Stefan, 2001 Antibody Engineering, Springer Laboratory Manuals; thedisclosures of which are incorporated herein by reference.

Alternatively, a library of PCSK9-specific antagonists in accordancewith the present invention may be brought into contact with PCSK9, andones able to demonstrate specific binding selected. Functional studiescan then be carried out to ensure proper functionality, e.g., inhibitionof PCSK9-dependent inhibition of cellular LDL uptake. There are varioustechniques available to the skilled artisan for the selection ofprotein-specific molecules from libraries using enrichment technologiesincluding, but not limited to, phage display (e.g., see technology fromCambridge Antibody Technology (“CAT”) disclosed in U.S. Pat. Nos.5,565,332; 5,733,743; 5,871,907; 5,872,215; 5,885,793; 5,962,255;6,140,471; 6,225,447; 6,291,650; 6,492,160; 6,521,404; 6,544,731;6,555,313; 6,582,915; 6,593,081, as well as other U.S. family membersand/or applications which rely on priority filing GB 9206318, filed May24, 1992; see also Vaughn et al., 1996, Nature Biotechnology14:309-314), ribosome display (see, e.g., Hanes and Pluckthün, 1997Proc. Natl. Acad. Sci. 94:4937-4942), bacterial display (see, e.g.,Georgiou, et al., 1997 Nature Biotechnology 15:29-34) and/or yeastdisplay (see, e.g., Kieke, et al., 1997 Protein Engineering10:1303-1310); the preceding disclosures of which are incorporatedherein by reference. A library, for example, can be displayed on thesurface of bacteriophage particles, with nucleic acid encoding thePCSK9-specific antagonist or fragment thereof expressed and displayed onits surface. Nucleic acid may then be isolated from bacteriophageparticles exhibiting the desired level of activity and the nucleic acidused in the development of desired antagonist. Phage display has beenthoroughly described in the literature; see, e.g., Konterrnann & Stefan,supra, and International Application Number WO 92/01047; the disclosuresof which are incorporated herein by reference. Specifically with regardto antibody molecules, individual heavy or light chain clones inaccordance with the present invention may also be used to screen forcomplementary heavy or light chains, respectively, capable ofinteraction therewith to form a molecule of the combined heavy and lightchains; see, e.g., International Application Number WO 92/01047. Anymethod of panning which is available to the skilled artisan may be usedto identify PCSK9-specific antagonists. Another specific method foraccomplishing this is to pan against the target antigen in solution,e.g. biotinylated, soluble PCSK9, and then capture the PCSK9-specificantagonist-phage complexes on streptavidin-coated magnetic beads, whichare then washed to remove nonspecifically-bound phage. The capturedphage can then be recovered from the beads in the same way they would berecovered from the surface of a plate, (e.g. DTT) as described herein.

PCSK9-specific antagonists may be purified by techniques available toone of skill in the art. Titers of the relevant antagonist preparation,ascites, hybridoma culture fluids, or relevant sample may be determinedby various serological or immunological assays which include, but arenot limited to, precipitation, passive agglutination, enzyme-linkedimmunosorbent antibody (“ELISA”) techniques and radioimmunoassay (“RIA”)techniques.

The present invention relates in part to methods employingPCSK9-specific antagonists described herein for antagonizing PCSK9function; said methods of which are further described below. Use of theterm “antagonizing” throughout the present application refers to the actof opposing, inhibiting, counteracting, neutralizing or curtailing oneor more functions of PCSK9. Inhibition or antagonism of one or more ofPCSK9-associated functional properties can be readily determinedaccording to methodologies known to the art (see, e.g., Barak & Webb,1981 J. Cell Biol. 90:595-604; Stephan & Yurachek, 1993 J. Lipid Res.34:325330; and McNamara et al., 2006 Clinica Chimica Acta 369:158-167)as well as those described herein. Inhibition or antagonism willeffectuate a decrease in PCSK9 activity relative to that seen in theabsence of the antagonist or, for example, that seen when a controlantagonist of irrelevant specificity is present. Preferably, aPCSK9-specific antagonist in accordance with the present inventionantagonizes PCSK9 functioning to the point that there is a decrease ofat least 10%, of the measured parameter including but not limited to theactivities disclosed herein, and more preferably, a decrease of at least20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 95% of the measuredparameter. Such inhibition/antagonism of PCSK9 functioning isparticularly effective in those instances where PCSK9 functioning iscontributing at least in part to a particular phenotype, disease,disorder or condition which is negatively impacting the subject.

In one aspect, the present invention provides a method for antagonizingthe activity of PCSK9, which comprises contacting a cell, population ofcells or tissue sample capable of being affected by PCSK9 (i.e., whichexpresses and/or comprises LDL receptors) with a PCSK9-specificantagonist disclosed herein under conditions that allow said antagonistto bind to PCSK9 when present and inhibit PCSK9's inhibition of cellularLDL uptake. Specific embodiments of the present invention include suchmethods wherein the cell is a human cell. Additional embodiments of thepresent invention include such methods wherein the cell is a murinecell.

In another aspect, the present invention provides a method forantagonizing the activity of PCSK9 in a subject, which comprisesadministering to the subject a therapeutically effective amount of aPCSK9-specific antagonist of the present invention. In specificembodiments, the methods for antagonizing PCSK9 function are for thetreatment of a PCSK9-associated disease, disorder or condition or,alternatively, a disease, disorder or condition that could benefit fromthe effects of a PCSK9 antagonist. The medicament would be useful in asubject(s) exhibiting a condition associated with PCSK9 activity, or acondition where the functioning of PCSK9 is contraindicated for aparticular subject. In select embodiments, the condition may behypercholesterolemia, coronary heart disease, metabolic syndrome, acutecoronary syndrome or related conditions.

The present invention, thus, contemplates the use of PCSK9-specificantagonists described herein in various methods of treatment whereantagonizing PCSK9 function is desirable. The method of treatment can beprophylactic or therapeutic in nature. In specific embodiments, thepresent invention relates to a method of treatment for a conditionassociated with/attributed to PCSK9 activity, or a condition where thefunctioning of PCSK9 is contraindicated for a particular subject, whichcomprises administering to the subject a therapeutically effectiveamount of a PCSK9-specific antagonist of the present invention. Inselect embodiments, the condition may be hypercholesterolemia, coronaryheart disease, metabolic syndrome, acute coronary syndrome or relatedconditions.

Methods of treatment in accordance with the present invention compriseadministering to an individual a therapeutically (or prophylactically)effective amount of a PCSK9-specific antagonist of the presentinvention. Use of the terms “therapeutically effective” or“prophylactically effective” in reference to an amount refers to theamount necessary at the intended dosage to achieve the desiredtherapeutic/prophylactic effect for the period of time desired. Thedesired effect may be, for example, amelioration of at least one symptomassociated with the treated condition. These amounts will vary, as theskilled artisan will appreciate, according to various factors, includingbut not limited to the disease state, age, sex and weight of theindividual, and the ability of the PCSK9-specific antagonist to elicitthe desired effect in the individual. The response may be documented byin vitro assay, in vivo non-human animal studies, and/or furthersupported from clinical trials.

The PCSK9-specific antagonist may be administered as a pharmaceuticalcomposition. The present invention, thus, provides a pharmaceuticallyacceptable composition comprising a PCSK9-specific antagonist of theinvention and a pharmaceutically acceptable carrier including but notlimited to an excipient, diluent, stabilizer, buffer, or alternativedesigned to facilitate administration of the antagonist in the desiredformat and amount to the treated individual.

The pharmaceutical composition may be formulated by any number ofstrategies known in the art, see, e.g., McGoff and Scher, 2000 SolutionFormulation of Proteins/Peptides: In—McNally, E. J., ed. ProteinFormulation and Delivery. New York, N.Y.: Marcel Dekker; pp. 139-158;Akers & Defilippis, 2000, Peptides and Proteins as Parenteral Solutions.In—Pharmaceutical Formulation Development of Peptides and Proteins.Philadelphia, Pa.: Taylor and Francis; pp. 145-177; Akers et al., 2002,Pharm. Biotechnol. 14:47-127. A pharmaceutically acceptable compositionsuitable for patient administration will contain an effective amount ofthe PCSK9-specific antagonist in a formulation which both retainsbiological activity while also promoting maximal stability duringstorage within an acceptable temperature range.

The antagonist-based pharmaceutically acceptable composition may, inparticular embodiments, be in liquid or solid form, or in the form ofgas particles or aerosolized particles. Any technique for production ofliquid or solid formulations may be utilized. Such techniques are wellwithin the realm of the abilities of the skilled artisan. Solidformulations may be produced by any available method including, but notlimited to, lyophilization, spray drying, or drying by supercriticalfluid technology. Solid formulations for oral administration may be inany form rendering the antagonist accessible to the patient in theprescribed amount and within the prescribed period of time. The oralformulation can take the form of a number of solid formulationsincluding, but not limited to, a tablet, capsule, or powder. Solidformulations may alternatively be lyophilized and brought into solutionprior to administration for either single or multiple dosing accordingto methods well known to the skilled artisan. Antagonist compositionsshould generally be formulated within a biologically relevant pH rangeand may be buffered to maintain a proper pH range during storage. Bothliquid and solid formulations generally require storage at lowertemperatures (e.g., 2-8° C.) in order to retain stability for longerperiods. Formulated antagonist compositions, especially liquidformulations, may contain a bacteriostat to prevent or minimizeproteolysis during storage, including but not limited to effectiveconcentrations (e.g., ≦1% w/v) of benzyl alcohol, phenol, m-cresol,chlorobutanol, methylparaben, and/or propylparaben. A bacteriostat maybe contraindicated for some patients. Therefore, a lyophilizedformulation may be reconstituted in a solution either containing or notcontaining such a component. Additional components may be added toeither a buffered liquid or solid antagonist formulation, including butnot limited to sugars as a cryoprotectant (including but not limited topolyhydroxy hydrocarbons such as sorbitol, mannitol, glycerol, anddulcitol and/or disaccharides such as sucrose, lactose, maltose, ortrehalose) and, in some instances, a relevant salt (including but notlimited to NaCl, KCl, or LiCl). Such antagonist formulations, especiallyliquid formulations slated for long term storage, will rely on a usefulrange of total osmolarity to both promote long term stability attemperatures of, for example, 2-8° C. or higher, while also making theformulation useful for parenteral injection. As appropriate,preservatives, stabilizers, buffers, antioxidants and/or other additivesmay be included. The formulations may contain a divalent cation(including but not limited to MgCl2, CaCl2, and MnCl2); and/or anon-ionic surfactant (including but not limited to Polysorbate-80 (Tween80™), Polysorbate-60 (Tween 60™), Polysorbate-40 (Tween 40™), andPolysorbate-20 (Tween 20™), polyoxyethylene alkyl ethers, including butnot limited to Brij 58™, Brij35™, as well as others such as TritonX-100™, Triton X-114™, NP40™, Span 85 and the Pluronic series ofnon-ionic surfactants (e.g., Pluronic 121)). Any combination of suchcomponents form specific embodiments of the present invention.

Pharmaceutical compositions in liquid format may include a liquidcarrier, e.g., water, petroleum, animal oil, vegetable oil, mineral oil,or synthetic oil. The liquid format may also include physiologicalsaline solution, dextrose or other saccharide solution or glycols, suchas ethylene glycol, propylene glycol or polyethylene glycol.

Preferably, the pharmaceutical composition may be in the form of aparenterally acceptable aqueous solution that is pyrogen-free withsuitable pH, tonicity, and stability. Pharmaceutical compositions may beformulated for administration after dilution in isotonic vehicles, forexample, Sodium Chloride Injection, Ringer's Injection, or LactatedRinger's Injection.

One aspect of the present invention is a pharmaceutical compositionwhich comprises: (i) about 50 to about 200 mg/mL of a PCSK9-specificantagonist described herein; (ii) a polyhydroxy hydrocarbon (includingbut not limited to sorbitol, mannitol, glycerol and dulcitol) and/or adisaccharide (including but not limited to sucrose, lactose, maltose andtrehalose); the total of said polyhydroxy hydrocarbon and/ordisaccharide being about 1% to about 6% weight per volume (“w/v”) of theformulation; (iii) about 5 mM to about 200 mM of histidine, imidazole,phosphate or acetic acid which serves as a buffering agent to prevent pHdrift over the shelf life of the pharmaceutical composition and as atonicity modifier; (iv) about 5 mM to about 200 mM of arginine, proline,phenylalanine, alanine, glycine, lysine, glutamic acid, aspartic acid ormethionine to counteract aggregation; (v) about 0.01M to about 0.1M ofhydrochloric acid (“HCl”) in an amount sufficient to achieve a pH in therange of about 5.5 to about 7.5; and (vi) a liquid carrier including butnot limited to sterile water, petroleum, animal oil, vegetable oil,mineral oil, synthetic oil, physiological saline solution, dextrose orother saccharide solution or glycols, such as ethylene glycol, propyleneglycol or polyethylene glycol; wherein said pharmaceutical compositionhas a pH in the range of about 5.5 to about 7.5; and wherein saidpharmaceutical composition optionally comprises about 0.01% to about 1%w/v of the formulation of a non-ionic surfactant (including but notlimited to Polysorbate-80 (Tween 80™), Polysorbate-60 (Tween 60™),Polysorbate-40 (Tween 40™), and Polysorbate-20 (Tween 20™),polyoxyethylene alkyl ethers, including but not limited to Brij 58™,Brij35™, as well as others such as Triton X-100™, Triton X-114™, NP40™,Span 85 and the Pluronic series of non-ionic surfactants (e.g., Pluronic121)).

HCl may be added as free acid, Histidine-HCl or Arginine-HCl. Wheresupplied as Histidine-HCl or Arginine-HCl, the total amounts ofHistidine or Arginine in the HCl form should be that specified above.Accordingly, some or all of the HCl depending on the amounts ofHistidine and/or Arginine may be supplied as Histidine-HCl and/orArginine-HCl; as appropriate. Use of the term “about” with respect toamounts disclosed in the specification means within 10% of the specifiednumbers provided. A range provided as, for example” in “about 50 toabout 200” expressly includes as distinct embodiments each number withinsaid range. As such in the above example, embodiments including but notlimited to those having 50, 100, 125, 150 and 200 form specificembodiments herein. Pharmaceutical compositions as disclosed herein havegeneral applicability despite the mode of administration. In specificembodiments, the disclosed pharmaceutical compositions are useful forsubcutaneous administration as a liquid or upon reconstitution of alyophilized form. In specific embodiments, PCSK9-specific antagonistsemployed in the disclosed formulations may be pegylated or form part offusion proteins.

Specific aspects of the present invention relate to the above disclosedpharmaceutical compositions which comprise: (i) about 50 to about 200mg/mL of a PCSK9-specific antagonist described herein; (ii) about 1% toabout 6% (in particular embodiments from about 2% to about 6%) w/vmannitol, trehalose or sucrose; (iii) about 10 mM to about 100 mM ofhistidine; (iv) about 25 mM to about 100 mM of arginine or proline; (v)about 0.02 M to about 0.05M of hydrochloric acid (“HCl”) in an amountsufficient to achieve a pH in the range of about 5.8 to about 7; and(vi) a liquid carrier including but not limited to sterile water,petroleum, animal oil, vegetable oil, mineral oil, synthetic oil,physiological saline solution, dextrose or other saccharide solution orglycols, such as ethylene glycol, propylene glycol or polyethyleneglycol; wherein said pharmaceutical composition has a pH in the range ofabout 5.8 to about 7; and wherein said pharmaceutical compositionoptionally comprising about 0.01% to about 1% w/v of the formulation ofa non-ionic surfactant (including but not limited to Polysorbate-80(Tween 80™), Polysorbate-60 (Tween 60™), Polysorbate-40 (Tween 40™), andPolysorbate-20 (Tween 20™), polyoxyethylene alkyl ethers, including butnot limited to Brij 58™, Brij35™, as well as others such as TritonX-100™, Triton X-114™, NP40™, Span 85 and the Pluronic series ofnon-ionic surfactants (e.g., Pluronic 121)).

Specific embodiments provide pharmaceutical compositions which comprise:(i) 50 to 200 mg/mL of a PCSK9-specific antagonist described herein;(ii) about 1% to about 6% (in particular embodiments from about 2% toabout 6%) w/v mannitol, trehalose or sucrose; (iii) about 10 mM to about150 mM of histidine; (iv) about 10 mM to about 150 mM of arginine orproline; (v) about 0.03 M to about 0.05 M of hydrochloric acid (“HCl”)in an amount sufficient to achieve a pH in the range of about 5.8 toabout 6.5; and (vi) a liquid carrier including but not limited tosterile water, petroleum, animal oil, vegetable oil, mineral oil,synthetic oil, physiological saline solution, dextrose or othersaccharide solution or glycols, such as ethylene glycol, propyleneglycol or polyethylene glycol; wherein said pharmaceutical compositionhas a pH in the range of about 5.8 to about 6.5; and wherein saidpharmaceutical composition optionally comprising about 0.01% to about 1%w/v of Polysorbate-80 (Tween 80™) or Polysorbate-20 (Tween 20™).

Specific embodiments herein provide pharmaceutical compositions whichcomprise: (i) 50 to 200 mg/mL of a PCSK9-specific antagonist describedherein; (ii) about 1% to about 6% (in particular embodiments from about2% to about 6%) w/v sucrose; (iii) about 25 mM to about 100 mM ofhistidine; (iv) about 25 mM to about 100 mM of arginine; (v) about 0.040M to about 0.045 M of hydrochloric acid (“HCl”) in an amount sufficientto achieve a pH of about 6; and (vi) sterile water; wherein saidpharmaceutical composition has a pH of about 6; and wherein saidpharmaceutical composition optionally comprising about 0.01% to about 1%w/v of Polysorbate-80 (Tween 80™) or Polysorbate-20 (Tween 20™). Inspecific embodiments thereof, the levels of histidine and arginine arewithin 25 mM of each other and, in other embodiments are the same.

Specific embodiments herein provide pharmaceutical compositions whichcomprise (i) 50 to 200 mg/mL of a PCSK9-specific antagonist describedherein; (ii) sucrose, histidine and arginine in one of the followingamounts: (a) about 1% w/v sucrose, about 10 mM histidine and about 25 mMarginine; (b) about 2% w/v sucrose, about 25 mM histidine and about 25mM arginine; (c) about 3% w/v sucrose, about 50 mM histidine and about50 mM arginine; or (d) about 6% w/v sucrose, about 100 mM histidine andabout 100 mM arginine; (iii) about 0.04 mol or, alternatively, about1.46 g of HCl; and (iv) sterile water; wherein said pharmaceuticalcomposition has a pH of about 6; and wherein said pharmaceuticalcomposition optionally comprising about 0.01% to about 1% w/v ofPolysorbate-80 (Tween 80™) or Polysorbate-20 (Tween 20™). Specificembodiments herein are wherein the amounts of sucrose, histidine andarginine in (ii) above are that described in (c) or (d).

Specific embodiments herein provide pharmaceutical compositions asdescribed which comprise 50 to 200 mg/ml of any one of the variousPCSK9-specific antagonists described herein. For purposes ofexemplification of one distinct embodiment thereof, and not to beconstrued as a limitation, is the following: a pharmaceuticalformulation as described above which comprises: a PCSK9-specificantagonist which comprises: (a) a light chain comprising SEQ ID NO: 26;and (b) a heavy chain comprising SEQ ID NO: 25; wherein saidPCSK9-specific antagonist is an antibody molecule that antagonizesPCSK9's inhibition of cellular LDL uptake.

Particular embodiments herein are pharmaceutical compositions accordingto the above description which are lyophilized and reconstituted. Inspecific embodiments, said protein concentration in said lyophilized andreconstituted solution is up to 2-fold higher than in thepre-lyophilized composition. In specific embodiments, the protein orPCSK9-specific antagonist concentration in the lyophilized and/orreconstituted pharmaceutical composition is in the range of about 50mg/mL to about 300 mg/mL Diluents useful for reconstituting thelyophilized pharmaceutical compositions include but are not limited tosterile water, bacterio static water for injection (“BWFI”),phosphate-buffered saline, a sterile saline solution, physiologicalsaline solution, Ringer's solution or dextrose solution and may inspecific embodiments contain 0.01-1% (w/v) of Polysorbate-80 (Tween 80™)or Polysorbate-20 (Tween 20™). In specific embodiments, lyophilizedpowder can be reconstituted with 1/60.2× original volume (or 0.167 mL)up to 1× (1 mL).

Exemplary embodiments of the present invention are pharmaceuticalcompositions as described herein which are stable. Other embodiments ofthe present invention are pharmaceutical compositions as describedherein which are stable to lyophilization and reconstitution. Variousmethods are available to the skilled artisan to prepare lyophilizedcompositions; see, e.g., Martin & Mo, 2007 “Stability Considerations forLyophilized Biologics” Amer. Pharm. Rev. “Stable” as used herein refersto the property of the protein or PCSK9-specific antagonist to retainits physical or chemical stability, conformational integrity, or itsability to exhibit less denaturation, protein clipping, aggregation,fragmentation, acidic variant formation or loss of biological activitycompared with a control sample at a temperature in the range of 4-37° C.for at least about 30 days. Other embodiments remain stable for up to 3months, 6 months, 12 months, 2 years or longer periods at the abovetemperatures. In specific embodiments the formulation exhibits nosignificant changes at 2-8° C. for at least 6 months, and preferably 12months, 2 years or longer, in order of preference. Specific embodimentsexperience less than 10% or, in particular embodiments, less than 5% ofdenaturation, protein clipping, aggregation, fragmentation, acidicvariant formation or loss of biological activity compared with a controlsample at a temperature in the range of 25-45° C. (or alternatively 2-8°C.) for at least about 30 days, 3 months, 6 months, 12 months, 2 yearsor longer. Stability of the formulations can be tested via several meansknown to the skilled artisan including, but not limited to SizeExclusion Chromatography (SEC-HPLC) to measure aggregation andfragmentation, Dynamic Light Scattering (DLS) to measure particle sizeof concentrated samples, capillary SDS-PAGE to measure fragmentation andcapillary iso-electric focusing (cIEF) or cation exchange chromatography(“CEX”) to measure acidic variants formation. Techniques suitable forthe analysis of protein stability are well understood by those of skillin the art: see review in Peptide and Protein Drug Delivery, 247-301,Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) andJones, 1993 Adv. Drug Delivery Rev. 10:29-90.

Pharmaceutical compositions as described herein should be sterile. Thereare various techniques available to the skilled artisan to accomplishthis including, but not limited to, filtration through sterilefiltration membranes. In specific embodiments, employing lyophilized andreconstituted compositions, this may be done prior to or followinglyophilization and reconstitution.

Dosing of antagonist therapeutics is well within the realm of theskilled artisan, see, e.g., Lederman et al., 1991 Int. J. Cancer47:659-664; Bagshawe et al., 1991 Antibody, Immunoconjugates andRadiopharmaceuticals 4:915-922, and will vary based on a number offactors including but not limited to the particular PCSK9-specificantagonist utilized, the patient being treated, the condition of thepatient, the area being treated, the route of administration, and thetreatment desired. A physician or veterinarian of ordinary skill canreadily determine and prescribe the effective therapeutic amount of theantagonist. Dosage ranges may be from about 0.01 to 100 mg/kg, and moreusually 0.05 to 25 mg/kg, of the host body weight. For example, dosagescan be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight,5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10mg/kg. For purposes of illustration, and not limitation, in specificembodiments, a dose of 5 mg to 2.0 g may be utilized to deliver theantagonist systemically. Optimal precision in achieving concentrationsof antagonist within a range that yields efficacy without toxicityrequires a regimen based on the kinetics of the drug's availability tothe target site(s). This involves a consideration of the distribution,equilibrium, and elimination of the PCSK9-specific antagonist.Antagonists described herein may be used alone at appropriate dosages.Alternatively, co-administration or sequential administration of otheragents may be desirable. It will be possible to present a therapeuticdosing regime for the PCSK9-specific antagonists of the presentinvention in conjunction with alternative treatment regimes. Forexample, PCSK9-specific antagonists may be used in combination or inconjunction with other drugs (therapeutic and/or prophylactic),including but not limited to cholesterol-lowering drugs, for example,cholesterol absorption inhibitors (e.g., Zetia®) and cholesterolsynthesis inhibitors (e.g., Zocor® and Vytorin®). The present inventioncontemplates such combinations and they form an important embodimenthereof. Accordingly, the present invention relates to methods oftreatment as described above where the PCSK9-specific antagonist isadministered/delivered simultaneously with, following or prior toanother drug or drugs (therapeutic and/or prophylactic), including butnot limited to cholesterol-lowering drugs, cholesterol absorportioninhibitors and cholesterol absorption inhibitors.

Individuals (subjects) capable of treatment as described herein includeprimates, human and non-human, and include any non-human mammal orvertebrate of commercial or domestic veterinary importance.

The PCSK9-specific antagonist may be administered to an individual byany route of administration appreciated in the art, including but notlimited to oral administration, administration by injection (specificembodiments of which include intravenous, subcutaneous, intraperitonealor intramuscular injection), administration by inhalation, intranasal,or topical administration, either alone or in combination with otheragents designed to assist in the treatment of the individual. ThePCSK9-specific antagonist may also be administered by injection devices,injector pens, needleless devices; and subcutaneous patch deliverysystems. The route of administration should be determined based on anumber of considerations appreciated by the skilled artisan including,but not limited to, the desired physiochemical characteristics of thetreatment. Treatment may be provided on a daily, weekly, biweekly, ormonthly basis, or any other regimen that delivers the appropriate amountof PCSK9-specific antagonist to the individual at the prescribed timessuch that the desired treatment is effected and maintained. Theformulations may be administered in a single dose or in more than onedose at separate times.

Also contemplated are methods of using the disclosed antagonists in themanufacture of a medicament for treatment of a PCSK9-associated disease,disorder or condition or, alternatively, a disease, disorder orcondition that could benefit from the effects of a PCSK9 antagonist. Themedicament would be useful in a subject(s) exhibiting a conditionassociated with PCSK9 activity, or a condition where the functioning ofPCSK9 is contraindicated ft. a particular subject. In selectembodiments, the condition may be hypercholesterolemia, coronary heartdisease, metabolic syndrome, acute coronary syndrome or relatedconditions.

PCSK9-specific antagonists disclosed herein may also be used as a methodof diagnosis of PCSK9. In select embodiments, the present inventionencompasses methods of identifying or quantifying the level of PCSK9present in a sample (including but not limited to a biological sample,e.g., serum or blood) which comprises contacting the sample with aPCSK9-specific antagonist described herein and detecting or quantifying,respectively, binding to PCSK9. The PCSK9-specific antagonist may beused in various assay formats known to the skilled artisan and may formpart of a kit (the general features of a kit of which are furtherdescribed below).

The present invention further provides for the administration ofdisclosed anti-PCSK9 antagonists for purposes of gene therapy. Throughsuch methods, cells of a subject are transformed with nucleic acidencoding a PCSK9-specific antagonist of the invention. Subjectscomprising the nucleic acids then produce the PCSK9-specific antagonistsendogenously. Previously, Alvarez, et al, Clinical Cancer Research6:3081-3087, 2000, introduced single-chain anti-ErbB2 antibodies tosubjects using a gene therapy approach. The methods disclosed byAlvarez, et al, supra, may be easily adapted for the introduction ofnucleic acids encoding an anti-PCSK9 antibody of the invention to asubject.

Nucleic acids encoding any PCSK9-specific antagonist may be introducedto a subject.

The nucleic acids may be introduced to the cells of a subject by anymeans known in the art. In preferred embodiments, the nucleic acids areintroduced as part of a viral vector. Examples of preferred viruses fromwhich the vectors may be derived include lentiviruses, herpes viruses,adenoviruses, adeno-associated viruses, vaccinia virus, baculovirus,alphavirus, influenza virus, and other recombinant viruses withdesirable cellular tropism.

Various companies produce viral vectors commercially, including, but byno means limited to, Avigen, Inc. (Alameda, Calif.; AAV vectors), CellGenesys (Foster City, Calif.; retroviral, adenoviral, AAV vectors, andlentiviral vectors), Clontech (retroviral and baculoviral vectors),Genovo, Inc. (Sharon Hill, Pa.; adenoviral and AAV vectors), Genvec(adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviralvectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpesviral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford,United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France;adenoviral, vaccinia, retroviral, and lentiviral vectors).

Methods for constructing and using viral vectors are known in the art(see, e.g., Miller, et al, BioTechniques 7:980-990, 1992). Preferably,the viral vectors are replication defective, that is, they are unable toreplicate autonomously, and thus are not infectious, in the target cell.Preferably, the replication defective virus is a minimal virus, i.e., itretains only the sequences of its genome which are necessary forencapsidating the genome to produce viral particles. Defective viruses,which entirely or almost entirely lack viral genes, are preferred. Useof defective viral vectors allows for administration to cells in aspecific, localized area, without concern that the vector can infectother cells. Thus, a specific tissue can be specifically targeted.

Examples of vectors comprising attenuated or defective DNA virussequences include, but are not limited to, a defective herpes virusvector (Kanno et al, Cancer Gen. Ther. 6:147-154, 1999; Kaplitt et al,J. Neurosci. Meth. 71:125-132, 1997 and Kaplitt et al, J. Neuro Onc.19:137-147, 1994).

Adenoviruses are eukaryotic DNA viruses that can be modified toefficiently deliver a nucleic acid of the invention to a variety of celltypes. Attenuated adenovirus vectors, such as the vector described byStrafford-Perricaudet et al, J. Clin. Invest. 90:626-630, 1992 aredesirable in some instances. Various replication defective adenovirusand minimum adenovirus vectors have been described (PCT Publication Nos.WO94/26914, WO94/28938, WO94/28152, WO94/12649, WO95/02697 andWO96/22378). The replication defective recombinant adenovirusesaccording to the invention can be prepared by any technique known to aperson skilled in the art (Levrero et al, Gene 101:195, 1991; EP 185573;Graham, EMBO J 3:2917, 1984; Graham et al, J. Gen. Virol. 36:59, 1977).

The adeno-associated viruses (AAV) are DNA viruses of relatively smallsize which can integrate, in a stable and site-specific manner, into thegenome of the cells which they infect. They are able to infect a widespectrum of cells without inducing any effects on cellular growth,morphology or differentiation, and they do not appear to be involved inhuman pathologies. The use of vectors derived from the AAVs fortransferring genes in vitro and in vivo has been described (see Daly, etal, Gene Ther. 8:1343-1346, 2001, Larson et al, Adv. Exp. Med. Bio.489:45-57, 2001; PCT Publication Nos. WO 91/18088 and WO 93/09239; U.S.Pat. Nos. 4,797,368 and 5,139,941 and EP 488528B1).

In another embodiment, the gene can be introduced in a retroviralvector, e.g., as described in U.S. Pat. Nos. 5,399,346, 4,650,764,4,980,289, and 5,124,263; Mann et al, Cell 33:153, 1983; Markowitz etal, J. Virol., 62:1120, 1988; EP 453242 and EP178220. The retrovirusesare integrating viruses which infect dividing cells.

Lentiviral vectors can be used as agents for the direct delivery andsustained expression of nucleic acids encoding a PCSK9-specificantagonist of the invention in several tissue types, including brain,retina, muscle, liver and blood. The vectors can efficiently transducedividing and nondividing cells in these tissues, and maintain long-termexpression of the PCSK9-specific antagonist. For a review, see Zuffereyet al, J. Virol. 72:9873-80, 1998 and Kafri et al, Curr. Opin. Mol.Ther. 3:316-326, 2001. Lentiviral packaging cell lines are available andknown generally in the art. They facilitate the production of high-titerlentivirus vectors for gene therapy. An example is atetracycline-inducible VSV-G pseudotyped lentivirus packaging cell linewhich can generate virus particles at titers greater than 10⁶ IU/ml forat least 3 to 4 days; see Kafri et al, J. Virol. 73:576-584, 1999. Thevector produced by the inducible cell line can be concentrated as neededfor efficiently transducing nondividing cells in vitro and in vivo.

Sindbis virus is a member of the alphavirus genus and has been studiedextensively since its discovery in various parts of the world beginningin 1953. Gene transduction based on alphavirus, particularly Sindbisvirus, has been well-studied in vitro (see Straus et al, Microbiol.Rev., 58:491-562, 1994; Bredenbeek et al, J. Virol., 67:6439-6446, 1993;Ijima et al, Int. J Cancer 80:110-118, 1999 and Sawai et al, Biochim.Biophyr. Res. Comm. 248:315-323, 1998. Many properties of alphavirusvectors make them a desirable alternative to other virus-derived vectorsystems being developed, including rapid engineering of expressionconstructs, production of high-titered stocks of infectious particles,infection of nondividing cells, and high levels of expression (Strausset al, 1994 supra). Use of Sindbis virus for gene therapy has beendescribed. (Wahlfors et al, Gene. Ther. 7:472-480, 2000 and Lundstrom,J. Recep. Sig. Transduct. Res. 19(1-4):673-686, 1999.

In another embodiment, a vector can be introduced to cells bylipofection or with other transfection facilitating agents (peptides,polymers, etc.). Synthetic cationic lipids can be used to prepareliposomes for in vivo and in vitro transfection of a gene encoding amarker (Feigner et al, Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987 andWang et al, Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987). Useful lipidcompounds and compositions for transfer of nucleic acids are describedin PCT Publication Nos. WO 95/18863 and WO 96/17823, and in U.S. Pat.No. 5,459,127.

It is also possible to introduce the vector in vivo as a naked DNAplasmid. Naked DNA vectors for gene therapy can be introduced intodesired host cells by methods known in the art, e.g., electroporation,microinjection, cell fusion, DEAE dextran, calcium phosphateprecipitation, use of a gene gun, or use of a DNA vector transporter(see, e.g., Wilson, et al, J. Biol. Chem. 267:963-967, 1992; Williams etal, Proc. Natl. Acad. Sci. USA 88:2726-2730, 1991). Other reagentscommonly used for transfection of plasmids include, but are by no meanslimited to, FuGene, Lipofectin, and Lipofectamine. Receptor-mediated DNAdelivery approaches can also be used (Wu et al, J. Biol. Chem.263:14621-14624, 1988). U.S. Pat. Nos. 5,580,859 and 5,589,466 disclosedelivery of exogenous DNA sequences, free of transfection facilitatingagents, in a mammal. Recently, a relatively low voltage, high efficiencyin vivo DNA transfer technique, termed electrotransfer, has beendescribed (Vilquin et al, Gene Ther. 8:1097, 2001; Payen et al, Exp.Hematol. 29:295-300, 2001; Mir, Bioelectrochemistiy 53:1-10, 2001; PCTPublication Nos. WO 99/01157, WO 99/01158 and WO 99/01175).

Pharmaceutical compositions suitable for such gene therapy approachesand comprising nucleic acids encoding an anti-PCSK9 antagonist of thepresent invention are included within the scope of the presentinvention.

In another aspect, the present invention provides a method foridentifying, isolating, quantifying or antagonizing PCSK9 in a sample ofinterest using a PCSK9-specific antagonist of the present invention. ThePCSK9-specific antagonists may be utilized as research tools inimmunochemical assays, such as Western blots, ELISAs, radioimmunoassay,immunohistochemical assays, immunoprecipitations, or otherimmunochemical assays known in the art (see, e.g., ImmunologicalTechniques Laboratory Manual, ed. Goers, J. 1993, Academic Press) orvarious purification protocols. The antagonists may have a labelincorporated therein or affixed thereto to facilitate readyidentification or measurement of the activities associated therewith.One skilled in the art is readily familiar with the various types ofdetectable labels (e.g., enzymes, dyes, or other suitable moleculeswhich are either readily detectable or cause some activity/result thatis readily detectable) which are or may be useful in the aboveprotocols.

An additional aspect of the present invention are kits comprisingPCSK9-specific antagonists or pharmaceutical compositions disclosedherein and instructions for use. Kits typically but need not include alabel indicating the intended use of the contents of the kit. The termlabel includes any writing, or recorded material supplied on or with thekit, or which otherwise accompanies the kit. In specific embodimentswherein the pharmaceutical composition is provided lyophilized, the kitmay include sterile water or saline for reconstitution of theformulation into liquid form. In specific embodiments, the amount ofwater or saline is from about 0.1 ml to 1.0 ml.

The following examples are provided to illustrate the present inventionwithout limiting the same hereto:

Example 1 Isolation of Recombinant Fab Display Phage

Recombinant Morphosys HuCAL Gold Fab phage display libraries (see, e.g.,Knappik et al., 2000 J. Mol. Biol. 296:57-86) were panned againstimmobilized recombinant murine PCSK9 through a process which is brieflydescribed as follows: Phage Fab display libraries were first dividedinto 3 pools: one pool of VH2+VH4+VH5, another of VH1+VH6, and a thirdpool of VH3. The phage pools and immobilized PCSK9 protein were blockedwith nonfat dry milk.

For the first round of panning, each phage pool was bound independentlyto V5-, His-tagged PCSK9 protein immobilized in wells of Nunc Maxisorpplate. Immobilized phage-PCSK9 complexes were washed sequentially with(1) PBS/0.5% Tween™ 20 (Three quick washes); (2) PBS/0.5% Tween™ 20 (One5 min. incubation with mild shaking); (3) PBS (Three quick washes); and(4) PBS (Two 5-min. incubations with mild shaking). Bound phages wereeluted with 20 mM DTT and all three eluted phage suspensions werecombined into one tube. E. coli TG1 were infected with eluted phages.Pooled culture of phagemid-bearing cells (chloramphenicol-resistant)were grown up, and frozen stock of phagemid-bearing culture were made.Phage were rescued from culture by co-infection with helper phage, andphage stock for next round of panning were made.

For the second round of panning, phages from Round 1 were bound toimmobilized, blocked V5-, His-tagged PCSK9 protein Immobilizedphage-PCSK9 complexes were washed sequentially with (1) PBS/0.05% Tween™20 (One quick wash); (2) PBS/0.05% Tween™ 20 (Four 5 min. incubationswith mild shaking); (3) PBS (One quick wash); and (4) PBS (Four 5-min.incubations with mild shaking). Bound phages were eluted, E. coli TG1cells were infected, and phage were rescued as in Round 1.

For the third round of panning, phages from Round 2 were bound toimmobilized, blocked V5-His-tagged PCSK9 protein. Immobilizedphage-PCSK9 complexes were washed sequentially with (1) PBS/0.05% Tween™20 (Ten quick washes); (2) PBS/0.05% Tween™ 20 (Five 5 min. incubationswith mild shaking); (3) PBS (Ten quick washes); and (4) PBS (Five 5-min.incubations with mild shaking). Bound phages were eluted and E. coli TG1cells were infected as in Round 1. Phagemid-infected cells were grownovernight and phagemid DNA was prepared.

XbaI-EcoRI inserts from Round 3 phagemid DNA were subcloned intoMorphosys Fab expression vector pMORPH_x9_MH to yield plasmid pMORPHx9MH/mPCSK9 2 CX1D05 (see, e.g., FIG. 1), and a library of Fab expressionclones was generated in E. coli TG1 F⁻. Transformants were spread onLB+chloramphenicol+glucose plates and grown overnight to generatebacterial colonies. Individual transformant colonies were picked andplaced into wells of two 96-well plates for growth and screening for Fabexpression.

Example 2 ELISA Screening of Bacterially Expressed Fabs

Cultures of individual transformants were IPTG-induced and grownovernight for Fab expression. Culture supernatants (candidate Fabs) wereincubated with purified V5-, His-tagged PCSK9 protein immobilized inwells of 96-well Nunc Maxisorp plates, washed with 0.1% Tween™ 20 in PBSusing a plate washer, incubated with HRP-coupled anti-Fab antibody, andwashed again with PBS/Tween™ 20. Bound HRP was detected by addition ofTMP substrate, and A₄₅₀ values of wells were read with a plate reader.

Negative controls were included as follows: Controls for nonspecific Fabbinding on each plate were incubated with parallel expressedpreparations of anti-EsB, an irrelevant Fab.

Growth medium only.

Positive controls for ELISA and Fab expression were included as follows:

EsB antigen was bound to three wells of the plate and subsequentlyincubated with anti-EsB Fab. To control for Fabs reacting with the V5 orHis tags of the recombinant PCSK9 antigen, parallel ELISAs wereperformed using V5-, His-tagged secreted alkaline phosphatase protein(SEAP) expressed in the same cells as the original PCSK9 antigen andsimilarly purified. Putative PCSK9-reactive Fabs were identified asyielding >3× background values when incubated with PCSK9 antigen butnegative when incubated with SEAP. Clones scoring as PCSK9-reactive inthe first round of screening were consolidated onto a single plate,re-grown in triplicate, re-induced with IPTG, and re-assayed in parallelELISAs vs. PCSK9 and SEAP. Positive and negative controls were includedas described above. Clones scoring positive in at least 2 of 3replicates were carried forward into subsequent characterizations. Incases of known or suspected mixed preliminary clones, cultures werere-purified by streaking for single colonies on 2×YT plates withchloramphenicol, and liquid cultures from three or more separatecolonies were assayed again by ELISAs in triplicate as described above.

Example 3 DNA Sequence Determination of PCSK9 ELISA-Positive Fab Clones

Bacterial culture for DNA preps was made by inoculating 1.2 ml 2×YTliquid media with chloramphenicol from master glycerol stocks ofpositive Fabs, and growing overnight. DNA was prepared from cell pelletscentrifuged out of the overnight cultures using the Qiagen Turbo Minipreps performed on a BioRobot 9600. ABI Dye Terminator cycle sequencingwas performed on the DNA with Morphosys defined sequencing primers andrun on an ABI 3100 Genetic Analyzer, to obtain the DNA sequence of theFab clones. DNA sequences were compared to each other to determineunique clone sequences and to determine light and heavy chain subtypesof the Fab clones.

Example 4 Expression and Purification of Fabs from Unique PCSK9ELISA-Positive Clone

Fabs from ELISA-positive clone m2CX1D05 and the EsB (negative control)Fab were expressed by IPTG-induction in E. coli TGIF⁻ cells. Cultureswere lysed and the His-tagged Fabs were purified by immobilized metalion affinity chromatography (IMAC), and proteins were exchanged into 25mM HEPES pH 7.3/150 mM NaCl by centrifugal diafiltration. Proteins wereanalyzed by electrophoresis on Caliper Lab-Chip 90 and by conventionalSDS-PAGE, and quantified by Bradford protein assay. Purified Fab proteinwas re-assayed by ELISA in serial dilutions to confirm activity ofpurified Fab. Positive and Negative controls were run as before.Purified Fab preparations were then analyzed as described below.

Example 5 Conversion of m2CX1D05 Fab to Full Length IgG

The DNA sequence encoding the m2CX1D05 light chain variable region wasamplified by polymerase chain reaction from plasmid templatepMORPHx9_MH/mPCSK9_(—)2_CX1_D05, using primers:ACAGATGCCAGATGCGATATCCAGATGACCCAGA (SEQ ID NO: 33) andTGCAGCCACCGTACGTTTAATTTCAACTTTCGTACC (SEQ ID NO: 34). The product ofthis amplification was cloned into plasmid pV1JNSA-GS-FB-LCK that hadbeen previously digested with FspI and BmtI, using the InFusion cloningsystem (Clontech). The resulting plasmid was verified by DNA sequencingacross the variable region. Endotoxin-free plasmid preparations weremade using the Qiagen Endo-Free plasmid maxiprep kit.

The DNA sequence encoding the heavy chain variable region ofpMORPHx9MH/mPCSK92_CX1_D05 was amplified by polymerase chain reactionusing primers: ACAGGTGTCCACTCGCAGGTGCAATTGGTTCAGTCT (SEQ ID NO: 35) andGCCCTTGGTGGATGCTGAGCTAACCGTCACCAGGGT (SEQ ID NO: 36), and the amplifiedproduct was cloned into plasmid pV1JNSA-BF-HCG2M4 that had beenpreviously digested with FspI and BmtI. The resulting plasmid wasverified by DNA sequencing across the variable region. Endotoxin-freeplasmid preparations were made using the Qiagen Endo-Free plasmidmaxiprep kit.

Full-length IgG was obtained by co-transfection of HEK293 cells with the1D05 light chain- and heavy-chain-encoding plasmids, following byProtein A purification of the expressed IgG.

Example 6 Kinetic Evaluation of Fab:PCSK9 Interactions with SurfacePlasmon Resonance (“Spr”)

SPR measurements were performed using a Biacore™ (Pharmacia BiosensorAB, Uppsala, Sweden) 2000 system. Sensor chip CM5 and Amine Coupling Kitfor immobilization were from Biacore™.

Anti-Fab IgG (Human specific) (Sigma, catalog #I5260) was covalentlycoupled to surfaces 1 and 2 of a Sensor Chip CM5 via primary aminegroups, using the immobilization wizard with the “Aim forimmobilization” option using Biacore™ Amine Coupling Kit (cat#BR-1000-50. A target immobilization of 5000 RU was specified. The wizarduses a 7 minute activation with a 1:1 mixture of 100 mM NHS(N-Hydroxysuccinimide) and 400 mM EDC(1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide), injects the ligand inseveral pulses to achieve the desired level, then deactivates theremaining surface with a 7 minute pulse of ethanolamine.

Anti-PCSK9 Fabs were captured on capture surface 2, and surface 1 wasused as a reference for kinetic studies of Fab:PCSK9 interactions. EachFab was captured by flowing a 500 ng/ml solution at 5 or 10 μl/min for1-1.5 minutes to reach a target R_(L) for an R_(max) of 100-150 RU forthe reaction. 5-10 concentrations of hPCSK9v5His or mPCSK9v5His antigenswere flowed across the surface at 30 μl/minute for 3-4 minutes. 15-60minutes dissociation time was allowed before regeneration of theAnti-Fab surface with a 30 second pulse of 10 mM glycine pH 2.0.

BiaEvaluation Software was used to evaluate the sensograms from themultiple concentration of PCSK9 antigen analyzed with each Fab, toestimate the kinetics constants of the Fab:PCSK9 interactions.

The kinetic constants were determined as follows:

TABLE 2 Fab Antigen k_(on) (1/Ms × 10⁻⁵⁾ k_(off) (1/s × 10⁴⁾ K_(D) (nM)m2CX1D05 Fab Human 0.22 ±0.01 2.47 ±0.05 11.5 ±0.75 mean (N = 3) PCSK9m2CX1D05 Fab Murine 0.86 ±0.02 2.57 ±0.19 3.35 ±0.39 mean (N = 3) PCSK9

Example 7 Kinetic Evaluation of IgG:PCSK9 Interactions with SurfacePlasmon Resonance (“Spr”)

SPR measurements were performed using a Biacore™ (Pharmacia BiosensorAB, Uppsala, Sweden) 2000 system. Sensor chip CM5 and Amine Coupling Kitfor immobilization were from Biacore™.

A goat Anti-Human IgG (Caltag, catalog #H10700) was covalently coupledto surfaces 1 and 2 of a Sensor Chip CM5 via primary amine groups, usingthe immobilization wizard with the “Aim for immobilization” option usingBiacore™ Amine Coupling Kit (cat# BR-1000-50. A target immobilization of5000 RU was specified. The wizard uses a 7 minute activation with a 1:1mixture of 100 mM NHS (N-Hydroxysuccinimide) and 400 mM EDC(1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide), injects the ligand inseveral pulses to achieve the desired level, then deactivates theremaining surface with a 7 minute pulse of ethanolamine.

Anti-PCSK9 IgGs were captured on capture surface 2, and surface 1 wasused as a reference for kinetic studies of IgG:PCSK9 interactions. IgGwas captured by flowing a 10 nM solution at 10 μl/min for 1-1.5 minutesto reach a target R_(L) for an R_(max) of 100-150 RU for the reaction.5-10 concentrations of hPCSK9v5His or mPCSK9v5His antigens were flowedacross the surface at 30 or 60 μl/minute for 4 minutes. 15-60 minutesdissociation time was allowed before regeneration of the Anti-IgGsurface with a 60 second pulse of 10 mM Glycine pH 1.7.

BiaEvaluation Software was used to evaluate the sensograms from themultiple concentration of PCSK9 antigen analyzed with each IgG, toestimate the kinetics constants of the IgG:PCSK9 interactions.

The kinetic constants were determined as follows:

TABLE 3 IgG Antigen k_(on) (1/Ms × 10⁻⁵⁾ k_(off) (1/s × 10⁴⁾ K_(D) (nM)1D05 IgG2m4 hPCSK9 0.88 ±0.01 3.16 ±0.27 3.6 ±0.33 mean (N = 2) 1D05IgG2m4 mPCSK9 0.67 ±0.06 2.15 ±0.16 3.2 ±0.06 mean (N = 2)

Example 8 PCSK9-LDLR TR-FRET Assay for 1D05

This assay is a variant of the one described in Fisher et al., 2007 J.Biol. Chem. 282:20502-20512. AlexaFluor647-labeled PCSK9 (finalconcentration 10 nM) was combined with varying amounts of 1D05 and tothis was added Eu(8044)-labeled LDLR ectodomain to a final concentrationof ˜1.5 nM (sufficient to give ˜20,000 counts at Fl₆₂₀ nM on theRubystar) in 10 mM HEPES (pH 7.4), 150 mM NaCl, 0.1 mM CaCl₂, 0.05%(w/v) BSA in a total volume of 50 μL using 96 well black Dynatech Ubottom plates. After at least 90 minutes of equilibration, samples wereread in a Rubystar reader (BMG Corp.) using 20 flashes per well, a 50usec integration delay, and a 200 usec total integration time. Data wereexpressed as the ratio of (Fl₆₆₅/Fl₆₂₀×10000) and an IC₅₀ for 1D05 wasdetermined from the inflection point of a sigmoidal dose-response curveusing a standard four parameter fit.

FIG. 2 illustrates the activity of 1D05 in the PCSK9-LDLR interactionTR-FRET assay. Both the Fab and IgG of 1D05 are potent and inhibit thePCSK9-LDLR interaction fully.

Example 9 Exopolar Assay: Effects of Exogenous PCSK9 on Cellular LDLUptake

On day 1, 30,000 HEK cells/well were plated in a 96 well polyD-lysinecoated plate. On day 2, the media was switched to no-serum containingDMEM media. On day 3, the media was removed and the cells were washedwith OptiMEM. Purified PCSK9 was added in 100 μl of DMEM mediacontaining LPDS and dI-LDL. The plates were incubated at 37° C. for 6.5hrs. The cells were washed quickly in TBS containing 2 mg/ml BSA; thenwashed in TBS-BSA for 2 minutes; and then washed twice (but quickly)with TBS. The cells were lysed in 100 μl RIPA buffer. Fluorescence wasthen measured in the plate using an Ex 520, Em 580 nm. The totalcellular protein in each well was measured using a BCA Protein Assay andthe fluorescence units were then normalized to total protein.

The Exopolar Assay is effective for characterizing variant effects onLDL uptake; see Table 4 below illustrating how the potencies of PCSK9mutants correlate with plasma LDL-cholesterol in the Exopolar Assay.

TABLE 4 EC-50 (nM) Mutation Gain/Loss LDL-C (mg/dI) Exopolar S127R Gain277 14 D374Y Gain 388 1.3 Wild-type 140 51 R46L Loss 116 78

Results:

m2CX1D05, both Fab and IgG, dose-dependently inhibited the effects ofboth human and murine PCSK9 on LDL uptake; an effect which wasreproducibly observed. The amount of PCSK9 added to the cells was˜100-320 nM.

m2CX1D05 (Fab) comprises a light chain of SEQ ID NO: 1 (comprising a VLof SEQ ID NO: 27) and a Fd chain of SEQ ID NO: 9 inclusive of linkersand tags (comprising a VH of SEQ ID NO: 11).

M2CX1D05 (IgG) comprises a light chain of SEQ ID NO: 26, and a heavychain comprising SEQ ID NO: 25.

FIGS. 3A-3D illustrate (i) 1D05 (Fab)'s dose-dependent inhibition ofmurine PCSK9-dependent loss of cellular LDL-uptake (FIG. 3A); (ii) 1D05(IgG)'s dose-dependent inhibition of murine PCSK9-dependent loss ofcellular LDL-uptake (FIG. 3B); (iii) 1D05 (Fab)'s dose-dependentinhibition of human PCSK9-dependent loss of cellular LDL-uptake (FIG.3C); and (iv) 1D05 (IgG)'s dose-dependent inhibition of humanPSCK9-dependent loss of cellular LDL-uptake (FIG. 3D).

1D05 clearly cross reacts with both human and mouse PCSK9. FIGS. 3A-3Dhave two controls: (i) a cell only control, showing the basal level ofcellular LDL uptake, and (ii) a cell+PCSK9 (5 μg/ml) control which showsthe level of PCSK9-dependent loss of LDL-uptake. The titrationexperiments which contain 1D05 and PCSK9 were done at a fixedconcentration of PCSK9 (5 μg/ml) and increasing concentrations of 1D05shown in the graphs.

1D05 can inhibit the effect of PCSK9 on cellular LDL uptake. IC₅₀s for1D05 (Fab) are 97 and 144 nM for mouse and human PCSK9 protein,respectively. IC₅₀s for 1D05 (IgG) are 85 and 79 nM for mouse and humanPCSK9 protein, respectively.

Example 10 PCSK9 Cellular Uptake

The assay that follows was carried out according to the methods ofFisher et al., 2007 J. Biol. Chem. 282: 20502-12.

Cells treated with Alexa Fluor 647-labeled PCSK9 were imaged as follows.CHO cells were plated on poly-D-lysine-coated 96-well optical CVGsterile black plates (Nunc) at a density of 20,000 cells/well. Cellswere plated in F-12K medium (nutrient mixture, Kaighn's modification(1×)) (Invitrogen) containing 100 units of penicillin and 100 μg/mlstreptomycin sulfate and supplemented with 10% FBS. Plates wereincubated overnight at 37° C. and 5% CO₂. The following morning, themedium was removed and replaced with 100 μl of F-12K medium containing100 units of penicillin and 100 μg/ml streptomycin sulfate. After 18 h,the medium was removed. Purified PCSK9 protein was labeled with AlexaFluor 647 as described under “Experimental Procedures.” Alexa Fluor647-labeled PCSK9 (1, 5, or 20 μg/ml) was added in 50 μl of F-12K mediumcontaining 10% lipoprotein-deficient serum to the cells. The plates wereincubated at 37° C. for 4 h, and the cells were washed quickly withTris-buffered saline before imaging. To label cellular nuclei, Hoechst33342 at a final concentration of 0.1 μg/ml was added to each well. Theplates were run on an Opera imager (Evotec Technologies GmbH, Hamburg,Germany) with a ×40 water immersion objective. Images were capturedusing excitation wavelengths of 405 nm for fluorescent nuclei and 635 nmfor Alexa Fluor 647-labeled PCSK9. For each well, 11 individual fieldscontaining >500 cells were captured for two emission wavelengths. Thedata were analyzed using a customized algorithm written using theAcapella language (Evotec Technologies GmbH). The algorithm identifiedand marked the nuclear and cytoplasmic areas of individual cells,followed by measurement of the total cytoplasmic intensity of the cell.The intensity was expressed in arbitrary fluorescent units.

For testing the 1D05 Ab, the identical procedure was used, but witheither HEK293 or HepG2 cells. For HepG2 cells, the plates would not havebeen poly-D-lysine coated. 5 μg/ml of AF647-labeled WT PCSK9 was addedalong with a titration of Fab ranging from 50 μg/ml down. Using thisprocedure, we obtained IC₅₀ values of roughly 80 nM for the Fab in bothcell types.

Results:

FIGS. 4A and 4B illustrate inhibition of PCSK9 internalization by theFab 1D05 and IgG 1D05 and restoration of LDL uptake.

Example 11 In Vivo Assay

Both Fab fragments and whole IgG of human 1D05 were tested in vivo inmice and changes in the level of LDL cholesterol were monitored. Themice used in these studies were (B6×B6-Tg(CETP) Ldlr^(tm1))F1 mice whichare hemizygous for the transgenic (Tg) expression of human CETP (whichmice lack) as well as the disruption of the LDL receptor (^(tm1)). Thesemice are particularly useful because of their human-like lipid profilesand LDL-rich nature.

Each mouse was bled twice, once at the beginning of the study toestablish individual baseline levels of LDL cholesterol (“pre”) and asecond time 3 hours later (“post”) to assess what changes took place inLDL levels after treatment. Each mouse received two IV doses ofDulbecco's PBS as a vehicle control, 1D05 IgG (0.5 mg), or 1D05 Fabfragments (0.5 mg) over the course of 3 hours. The 1D05 whole IgG wascentrifuged at 230,000×g to remove aggregates immediately prior toinjection.

In FIG. 5, the LDL levels for each mouse are represented by a set ofconnected symbols and the change in LDL (postbleed-prebleed) is shown asan average for each treatment group (A mg/dL). Treatment with PBS had noeffect on LDL measurements (−4 mg/dL, 5% reduction). In contrast, serumLDL was reduced 20% with 1D05 whole IgG (−19 mg/dL) and 34% with Fabfragments of 1D05 (−24 mg/dL).

Example 12 Limited Proteolysis

The limited proteolysis mass spectrometry strategy consists in theincubation of wt-hPCSK9 and 1D05/wt-hPCSK9 complex (substrates) withendoproteinase enzymes of different specificity in carefully controlledconditions (i.e., low enzymes concentration and short digestion time).Under these conditions, the endoproteases will cleave only the primarycleavage sites of the protein substrate (i.e., sites that are on thesurface of the protein substrate and exposed to the solvent). Thebinding of 1D05 Fab to wt-hPCSK9 will mask some surface residuesnormally exposed to the solvent in both proteins. Therefore the primarysites cleaved in wt-hPCSK9 and not in the 1D05/wt-hPCSK9 complexcorrespond to residues of PCSK9 protected by 1D05 in the complex. Someof these residues are likely to be directly involved in 1D05 binding.The proteolytic peptides generated by wt-hPCSK9 and 1D05/wt-hPCSK9limited proteolysis are identified and characterized by analysis of thedigest by Matrix Assisted Laser Desorption/Ionization Mass Spectrometry(MALDI-MS). Finally, the use of endoproteases with different specificityhelps to more accurately define the residues involved in binding.

The amount of proteolytic enzyme normally used in limited proteolysisexperiments had to be considerably reduced to avoid excess hydrolysis ofwt-hPCSK9 and loss of the primary binding sites (exposed residues).Incubation of wt-hPCSK9, 1D05 and 1D05/wt-hPCSK9 with endoproteases wasdone in 25 mM HEPES pH 7.5, 150 mM NaCl at room temperature. Theendoproteases used were AspN added at a 2500/1 (w/w) excess of proteincompared to proteolytic enzyme, and Trypsin and GluC added at a 1000/1(w/w) protein to endoprotease ratio. At periods of time 5, 15 and 30minutes after endoprotease addition, an aliquot of sample was depositedonto the MALDI target and subjected to direct MALDI-MS analysis in thepresence of sinapinic acid (SA) as matrix. The fragment peptidesoriginated from wt-PCSK9 after incubation with the proteolytic enzyme atvarious time were compared with those originated from wt-hPCSK9 in the1D05/wt-hPCSK9 complex sample to identify the residues protected fromproteolysis in the 1D05/wt-hPCSK9 interaction. The Figures and Tablesprovided herein report only the most relevant fragment peptides.

Limited Proteolysis with Endoprotease AspN:

The wt-hPCSK9 protein, 1D05/wt-hPCSK9 complex and 1D05 Fab wereincubated for 5, 15 and 30 minutes in the presence of AspN, whichcleaves N-terminally to Asp residues, at a 2500/1 (w/w) ratio betweenthe protein and the proteolytic enzyme. MALDI-MS analysis of the digestsrevealed the primary wt-hPCSK9 and 1D05/wt-hPCSK9 complex cleavage sites(see FIGS. 7A and 7B and Table 5 below). Table 5 illustrates thewt-hPCSK9 fragment peptides obtained after AspN incubation for 5 minuteswith wt-PCSK9 and 1D05/wt-hPCSK9 complex. In italics are peptides formedonly when wt-hPCSK9 hydrolyzes.

TABLE 5 Measured m/z Expected MW Peptide Cleaved AA 1969.1 1969.1153-168 Asp169 2222.0 2222.0 31-49 Asp49  4412.9 4411.2 698-737 Asp698

The species at m/z 1969.1, originated from the cleavage at Asp169 andmatching the theoretical mass of peptide 153-168 of the catalytic domainof wt-hPCSK9, was formed only in the wt-hPCSK9 sample indicating thatthis residue is protected from proteolysis by 1D05 Fab binding in the1D05/wt-hPCSK9 complex. Several species were formed in both wt-hPCSK9and 1D05/wt-hPCSK9 hydrolyses. In particular the ions at m/z 2222.0 and4412.9 corresponding to peptides 31-49 of the prodomain and 698-737 ofthe catalytic domain of wt-hPCSK9 were originated from cleavage at Asp49and Asp698.

At longer endoprotease AspN incubation time (i.e., 15, 30 minutes) thepeptide profile shown in the MALDI-MS spectra did not changesignificantly compared to the one at 5 minutes shown in FIGS. 7A and 7Bconfirming that Asp169 is protected from hydrolysis in the 1D05/wt-PCSK9interaction.

It is important to note that the observed degree of agreement betweenthe expected and measured mass values is within the norm for this typeof experiment, since mass calibration must be made with an externalstandard.

Limited Proteolysis with Endoprotease GluC:

Endoprotease GluC cleaves C-terminally Glu residues. Incubation of GluCwith wt-hPCSK9, 1D05/wt-hPCSK9 complex and 1D05 was conducted at a1000/1 and 100/1 (w/w) ratio between protein and proteolytic enzyme. Todetect the primary cleavage sites, the MALDI-MS analysis of the sampleswas conducted after 5, 15 and 30 minutes of incubation. The wt-hPCSK9residues cleaved in the wt-hPCSK9 protein and protected in the1D05/wt-hPCSK9 complex, and the corresponding peptides detected in theMS spectrum, are shown in FIGS. 8A-H and Table 6 below. Table 6illustrates peptide fragments obtained upon GluC incubation for 15minutes with wt-PCSK9 and 1D05/wt-PCSK9 complex. In italics are thepeptides originating from wt-PCSK9 and not from the complex.

TABLE 6 Measured m/z Expected MW Peptide Cleaved AA 1675.9 1675.8182-195 Glu181 Glu195 1918.0 1918.0 182-197 Glu181 Glu197 2213.2 2213.1153-170 Glu170 3357.4 3357.7 153-181 Glu181

With endoprotease GluC, protection is shown in the wt-hPCSK9 surfacearea including residues Glu170, Glu197 and Glu195. The specie at m/z3357.4, corresponding to peptide 153-181, and obtained in the incubationwith GluC of both wt-hPCSK9 and 1D05/wt-hPCSK9, indicates that Glu181 isnot protected by the Fab 1D05 binding to wt-hPCSK9.

Limited Proteolysis with Trypsin:

Trypsin cleaves C-terminally Arg and Lys residues. The enzyme was addedat 1000 (w/w) ratio to wt-PCSK9, 1D05/wt-PCSK9 complex and 1D05 for 5,15 and 30 minutes. MALDI-MS analysis of the wt-PCSK9 and 1D05/wt-PCSK9complex is shown in FIGS. 9A-D and the most relevant peptide fragmentsare reported in Table 7. Table 7 illustrates peptide fragments obtainedupon Trypsin incubation for 5 minutes with wt-PCSK9 and 1D05/wt-PCSK9complex. In italics are the peptides originating from wt-PCSK9 and notfrom the complex.

TABLE 7 Measured m/z Expected MW Peptide Cleaved AA 1877.9 1878.0 31-46Arg46  2279.3 2279.5 200-218 Arg199 Arg218 2581.9 2581.9 706-729 Arg705Arg729 3562.9 3562.9 706-737 Arg705 3909.9 3910.2 166-199 Arg165 Arg1994474.6 4474.9 161-199 Arg160 Arg199 5410.5 5410.8 168-215 Arg167 Arg2155729.7 5730.2 166-215 Arg165 Arg215 5850.8 5851.3 168-218 Arg167 Arg2186170.2 6170.7 166-218 Arg165 Arg218 7290.7 7291.0 153-215 Arg215 7731.57731.5 153-218 Arg218

The primary cleavage sites at 5 minutes were Arg46 on the prodomain andArg160, Arg165, Arg167, Arg199, Arg215, Arg218, Arg705 and Arg729 on thecatalytic domain of wt-hPCSK9. The species at m/z 2279.3, 3909.9 and4474.6 corresponding to peptides 200-218, 166-199 and 161-199 aredetected only in the wt-hPCSK9 hydrolysis and indicate that residueArg199 is protected by 1D05 binding. These peptide fragments togetherwith the species at m/z 5410.5, 5729.7, 5850.8, 6170.2 correspond topeptides 168-215, 166-215, 168-218, 166-218, detected only in wt-PCSK9thus indicating protection also on residues Arg165 and Arg167.

At 15 minutes of Trypsin incubation, protection on residue Arg199 isconfirmed. In fact the species at m/z 2280.0, 3591.4, 3910.9 and 4475.6,all originated from cleavage at Arg199, are present only in the wt-PCSK9hydrolysis and become more abundant. In addition, protection at Arg194is detected (as shown by the presence of the specie at m/z 3325.7 in thewt-PCSK9 spectrum). The species originated from cleavage at Arg165 andArg167 (ink 5411.5, 5730.9, 5851.8 and 6171.6) are present in thewt-hPCSK9 hydrolysis and start to appear with much lower intensity alsoin the 1D05/wt-hPCSK9 complex proteolysis. This may indicate that theprotection from proteolysis on such residues is due to steric hindranceof the Fab rather than to primary contacts between 1D05 and wt-PCSK9residues.

Results:

With LP-MS using three enzymes of different specificity, we identifiedthe surface area of wt-hPCSK9 protected by the 1D05 Fab in the1D05-wt-hPCSK9. Arg165, Arg167, Asp169, Glu170, Arg194, Glu197 andArg199 are the residues of wt-hPCSK9 protected upon binding to the 1D05Fab (see FIG. 10). These residues belong to the catalytic domain ofwt-hPCSK9 and are exposed on the surface of the molecule (see FIG. 11).In addition, the limited proteolysis with trypsin shows the possibilitythat residues 194-199 are directly involved in 1D05 binding whereasprotection from proteolysis on residues Arg165 and Arg167 may be due tosteric hindrance of the Fab instead of direct contacts between 1D05 andwt-PCSK9 residues.

Residues in peptides R194-R199 are conserved in human and mouse PCSK9.1D05 Fab recognizes human and mouse protein. As illustrated by thesequence alignment between human and mouse PCSK9 (see FIG. 12), theresidues included in the peptide 194-199 of wt-PCSK9 and protected by1D05 are conserved in both human and mouse PCSK9 while residues inpeptide 165-169 (also protected by 1D05 binding) are not. This wouldsupport a hypothesis that only residues 194-199 are directly interactingwith 1D05 while the others (165-169) are protected from proteolysis bysteric hindrance.

Example 13 PCSK9/1D05 TR-FRET Assay

Anti-V5 antibody (QED Biosciences) was labeled and purified as describedpreviously (see Fisher et al., 2007 J. Biol. Chem. 282 (28):20502-20512) using 4 equivalents of AlexaFluor 647 (Invitrogen). 1D05IgG was labeled in a similar manner using 5 equivalents of Eu(W8044)-DTA(Perkin-Elmer). Materials were protected from light and stored at 4° C.prior to use. V5/His-PCSK9 was generated as described previously (seeFisher et al., Id.).

TR-FRET assays were carried out in black Microfluor 2 96 well plates(Dynex Technologies) in 10 mM Hepes pH 7.4, 150 mM NaCl, 100 uM CaCl₂and 0.05% BSA. To 25 μL of 20 nM each AF647 labeled anti-V5 antibody andV5/His-PCSK9 was added a serial dilution of the unlabeled candidateantibody (i.e., 1D05 and 1B20), either Fab or IgG. Reagents wereequilibrated for ˜15 minutes at room temperature and then Eu(W8044)-1D05IgG was added to give a final concentration of 1.5 nM Eu labeledantibody (18000 counts at Fl₆₂₀ nm; S/B=12) and a total volume of 50 uL.After, equilibration assays were read in a BMG LabTech Rubystar Readeras described previously (Fisher et al., Id.). Data are reported asFl₆₆₅/Fl₆₂₀×10000. IC₅₀s were determined using data fitted to asigmoidal dose response curve using non-linear regression analysis(Kaleidagraph 4.03, Synergy Software).

FIG. 13 illustrates an analysis of 1D05 and a distinct antibody 1B20 ina PCSK9-1D05 interaction TR-FRET assay. Both Fabs are potent and inhibitthe interaction fully. FIGS. 14A-D illustrates 1B20's inhibition ofPCSK9 in the Exopolar assay described, e.g., in Example 9. 1B20 Fabinhibited murine PCSK9 at an IC₅₀ of 152 nM (n=5); and human PCSK9 at anIC₅₀ of 145 nM (n=5). 1B20 IgG inhibited murine PCSK9 at an IC₅₀ of 13nM; and human PCSK9 at an IC₅₀ of 22 nM. The binding particulars of 1B20Fab are illustrated in the following Table.

TABLE 8 hPCSK9v5His mPCSK9v5His k_(a) (1/Ms) 6.6E+04 ± 6.1E+03 1.41E+05± 1.2E+04 k_(d) (1/s) 4.8E−05 ± 7.4E−06 7.18E−05 ± 2.9E−06 K_(A) (1/M)1.5E+09 ± 3.0E+08  2.0E+09 ± 1.5E+08 K_(D) (M) 7.4E−10 ± 1.6E−10 5.1E−10 ± 3.8E−11

Example 14 1D05 Rhesus PK/PD Study

To characterize pharmacokinetics, pharmacodynamics and target engagementof 1D05, a single dose IV study was conducted in male Rhesus monkeys at3 mg/kg (7.0-9.0 kg, n=3). All Rhesus monkeys used in the study werenaïve to biologics.

Monkeys were given an IV bolus dose of 1D05 via the cephalic vein. Bloodsamples were collected from the saphenous/femoral vessel at designatedtime points post dosing and the resulting plasma/serum was stored at−70° C. until analysis.

The dosing solutions of 1D05 were prepared at 47.2 mg/mL in 100 mMHistidine, 100 mM Arginine, 6% sucrose, pH 6.0. The dosing solutionswere stored at 4° C. and kept on wet ice during dosing.

The lipoprotein analysis of the serum samples were carried out asdescribed below. An anti-human IgG ELISA using commercially availablereagents was used to quantify 1D05 levels.

As shown in FIG. 15, 1D05 lowered LDL-C by ˜50% at 3 mpk and ≧25% LDL-Clowering was observed for ˜16 days. The t_(1/2) of 1D05 (FIG. 16) was 77hr.

Example 15 Lipoprotein Analysis of Plasma/Serum Samples from 1D05 RhesusPK/PD Study

To generate lipoprotein profiles, plasma or serum was fractionated bychromatography over Superose-6 size exclusion column (GE LifeSciences,Inc.). Total cholesterol levels in the column effluent were continuouslymeasured via in-line mixture with a commercially available enzymaticcolorimetric cholesterol detection reagent (Total Cholesterol E, WakoUSA) followed by downstream spectrophotometric detection of the reactionproducts at 600 nm absorbance. The first peak of cholesterol eluted fromthe column was attributed to VLDL, the second peak to LDL and the thirdto HDL; the area under each peak was calculated using software providedwith the HPLC. To calculate the cholesterol concentration for eachlipoprotein fraction, the ratio of the corresponding peak area to totalpeak area was multiplied by the total cholesterol concentration measuredin the sample.

Example 16 Formulation

Monoclonal antibodies comprising a light chain comprising SEQ ID NO: 26and a heavy chain comprising SEQ ID NO: 25) were dialyzed into theappropriate formulations and concentrated. Solutions were then dispensedinto 3 mL glass vials for stability studies. Studies carried out inliquid form were immediately placed on stability at 2-8° C. or 25° C.

Analytical methods included Size Exclusion Chromatography (SEC-HPLC) tomeasure aggregation and fragmentation. Below is a table of Time 0 and 6MSEC data. The formulations containing 3/50/50 or 6/100/100(sucrose/His/Arg) form fewer aggregates and fragments after storage for6 months at 2-8° C. or 25° C. All formulations are at 6.0 except for thestandard—that is frozen at 1 mg/mL in Phosphate buffered saline (pH˜7).

TABLE 9 % High Order % % % Sample Name Aggregates Dimer Monomer Clipped1D05 standard T0 0.32% 1.59% 98.09% 0.00% 10His/150 NaCl Time 0 0.26%1.59% 98.15% 0.00% 3/50/50 Time 0 0.30% 1.59% 98.11% 0.00% 1D05 standard−70C 6M 0.60% 2.79% 96.61% 0.00% His/NaCl 4C 6M 0.98% 3.18% 95.83% 0.01%6/100/100 4C 6M 0.95% 2.90% 96.13% 0.02% 3/50/50 4C 6M 0.92% 3.00%96.07% 0.01% 100 mg/mL 4C 6M 0.97% 3.13% 95.85% 0.05% His/NaCl 25C 6M1.71% 4.45% 93.45% 0.40% 6/100/100 25C 6M 1.16% 3.69% 94.74% 0.39%3/50/50 25C 6M 1.31% 3.70% 94.62% 0.37%

Example 17 Variants

Site-directed mutant variants of 1D05 were generated and are disclosedherein as SEQ ID NOs: 51-60. Kds of site-directed mutant variants of1D05 Fabs were determined using a Bio-Rad ProteOn; with affinity beingmeasured against human PCSK9-V5-His. The methodologies for measuring Fabaffinities are essentially the same as previously described forBiacore®.

TABLE 10 Ab ID Chain Mutated Comprising VH KD (nM) H32Y HEAVY SEQ ID NO:51 2.01 M48AQ HEAVY SEQ ID NO: 52 2.06 M48L HEAVY SEQ ID NO: 53 1.52H99Y HEAVY SEQ ID NO: 54 1.45 M48L/M109L/M115L HEAVY SEQ ID NO: 55 1.13M48V HEAVY SEQ ID NO: 56 1.95 N50D LIGHT SEQ ID NO: 57 3.42 N50Q LIGHTSEQ ID NO: 58 0.615 N50T LIGHT SEQ ID NO: 59 2.13 N50Y LIGHT SEQ ID NO:60 2.58 * Amino acid numbering begins with the first residue of FR1,immediately following signal peptide.

What is claimed is:
 1. An isolated PCSK9-specific antagonist whichcomprises: (a) a heavy chain variable region comprising a CDR3 domaincomprising SEQ ID NO: 17 or an equivalent thereof, said equivalentcharacterized as having one or more conservative amino acidsubstitutions in the CDR3 domain; and/or (b) a light chain variableregion comprising a CDR3 domain comprising SEQ ID NO: 7 or an equivalentthereof, said equivalent characterized as having one or moreconservative amino acid substitutions in the CDR3 domain; wherein saidPCSK9-specific antagonist antagonizes PCSK9's inhibition of cellular LDLuptake.
 2. The PCSK9-specific antagonist of claim 1 wherein the CDR3domain(s) are in a human germline region in the CDR3 region thereof. 3.The PCSK9-specific antagonist of claim 1 that binds to human PCSK9 withan equilibrium dissociation constant (K_(D)) of less than 1200 nM. 4.The PCSK9-specific antagonist of claim 1 that binds to human PCSK9 witha K_(D) of less than 500 nM.
 5. The PCSK9-specific antagonist of claim 1that binds to human PCSK9 with a K_(D) of less than 100 nM.
 6. ThePCSK9-specific antagonist of claim 1 that binds to human PCSK9 with aK_(D) of less than 5 nM.
 7. The PCSK9-specific antagonist of claim 1that antagonizes PCSK9's inhibition of cellular LDL uptake at an IC₅₀ ofless than 500 nM.
 8. The PCSK9-specific antagonist of claim 1 thatantagonizes PCSK9's inhibition of cellular LDL uptake at an IC₅₀ of lessthan 200 nM.
 9. The PCSK9-specific antagonist of claim 1 thatantagonizes PCSK9's inhibition of cellular LDL uptake at an IC₅₀ of lessthan 100 nM.
 10. The PCSK9-specific antagonist of claim 1 thatantagonizes PCSK9's inhibition of cellular uptake by at least 20%. 11.The PCSK9-specific antagonist of claim 1 which is an antibody molecule.12. The PCSK9-specific antagonist of claim 1 which further comprises:(a) a heavy chain variable CDR1 sequence comprising SEQ ID NO: 13; (b) aheavy chain variable CDR2 sequence comprising SEQ ID NO: 15; (c) a lightchain variable CDR1 sequence comprising SEQ ID NO: 3; and/or (d) a lightchain variable CDR2 sequence comprising SEQ ID NO:
 5. 13. ThePCSK9-specific antagonist of claim 12 wherein the CDR1, CDR2 and/or CDR3domain(s) are in a human germline region in the respective CDR1, CDR2and/or CDR3 regions thereof.
 14. The PCSK9-specific antagonist of claim1 comprising: (a) a heavy chain variable region comprising a CDR3 domaincomprising SEQ ID NO: 17; (b) a light chain variable region comprising aCDR3 domain comprising SEQ ID NO: 7; (c) a heavy chain variable CDR1sequence comprising SEQ ID NO: 13; (d) a light chain variable CDR1sequence comprising SEQ ID NO: 3; (e) a heavy chain variable CDR2sequence comprising SEQ ID NO: 15; and (f) a light chain variable CDR2sequence comprising SEQ ID NO:
 5. 15. The PCSK9-specific antagonist ofclaim 14 wherein the CDR1, CDR2 and/or CDR3 domain(s) are in a humangermline variable region in the respective CDR1, CDR2 and/or CDR3regions thereof.
 16. The PCSK9-specific antagonist of claim 12 whichcomprises a heavy chain variable region comprising SEQ ID NO: 11 and/ora light chain variable region comprising SEQ ID NO:
 27. 17. ThePCSK9-specific antagonist of claim 12 which comprises a heavy chainhaving constant sequence comprising: SEQ ID NO:
 24. 18. ThePCSK9-specific antagonist of claim 16 which comprises a heavy chainhaving constant sequence comprising: SEQ ID NO:
 24. 19. A PCSK9-specificantagonist which comprises: (a) a light chain comprising SEQ ID NO: 1;and (b) a heavy chain comprising SEQ ID NO: 11; wherein saidPCSK9-specific antagonist is an antibody molecule that antagonizesPCSK9's inhibition of cellular LDL uptake.
 20. A PCSK9-specificantagonist of claim 19 wherein SEQ ID NO: 11 is followed in sequence byan amino acid sequence selected from the group consisting of: SEQ ID NO:21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO:
 24. 21. An isolatedPCSK9-specific antagonist which comprises: (a) a light chain comprisingSEQ ID NO: 26; and (b) a heavy chain comprising SEQ ID NO: 25; whereinsaid PCSK9-specific antagonist is an antibody molecule that antagonizesPCSK9's inhibition of cellular LDL uptake.
 22. An isolatedPCSK9-specific antagonist that: (a) inhibits the binding of a 1D05 Fabto PCSK9 by at least 50%; said 1D05 Fab characterized as comprising alight chain comprising SEQ ID NO: 1 and an Fd chain comprising aminoacids 1-233 of SEQ ID NO: 9; and (b) antagonizes (i) PCSK9 binding tothe LDL receptor and/or (ii) PCSK9 internalization into cells.
 23. APCSK9-specific antagonist that: (a) inhibits the binding of a 1D05 IgGto PCSK9 by at least 50%; said 1D05 IgG characterized as comprising (i)a light chain comprising SEQ ID NO: 1 and (ii) a heavy chain comprisingSEQ ID NO: 11; and (b) antagonizes (i) PCSK9 binding to the LDL receptorand/or (ii) PCSK9 internalization into cells.
 24. A PCSK9-specificantagonist of claim 23 wherein SEQ ID NO: 11 is followed in sequence byan amino acid sequence selected from the group consisting of: SEQ ID NO:21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO:
 24. 25. An isolatedPCSK9-specific antagonist which comprises: (a) heavy chain variableregion CDR3 sequence of SEQ ID NO: 45; (b) heavy chain variable regionCDR3 sequence of SEQ ID NO: 45; heavy chain CDR1 sequence of SEQ ID NO:43 and heavy chain CDR2 sequence of SEQ ID NO: 44; (c) light chainvariable region CDR3 sequence of SEQ ID NO: 48; (d) light chain variableregion CDR3 sequence of SEQ ID NO: 48; light chain CDR1 sequence of SEQID NO: 46 and light chain CDR2 sequence of SEQ ID NO: 47; (e) both (a)and (c); (f) both (b) and (d); (g) heavy and/or light chain variableregions comprising SEQ ID NOs: 50 and 49, respectively; (h) a heavychain variable region comprising any one of SEQ ID NOs: 51-56 andoptionally a light chain variable region comprising SEQ ID NO: 27; or(i) a light chain variable region comprising any one of SEQ ID NOs:57-60 and optionally a heavy chain variable region comprising SEQ ID NO:11; wherein said PCSK9-specific antagonist is an antibody molecule thatantagonizes PCSK9's inhibition of cellular LDL uptake.
 26. A compositioncomprising the PCSK9-specific antagonist of claim 1 and apharmaceutically acceptable carrier.
 27. A composition in accordancewith claim 26 which comprises: (a) about 50 mg/mL to about 200 mg/mL ofthe PCSK9-specific antagonist; (b) a polyhydroxy hydrocarbon (includingbut not limited to sorbitol, mannitol, glycerol and dulcitol) and/or adisaccharide (including but not limited to sucrose, lactose, maltose andtrehalose); the total of said polyhydroxy hydrocarbon and/ordisaccharide being about 1% to about 6% w/v of the formulation; (c)about 5 mM to about 200 mM of histidine, imidazole, phosphate or aceticacid; (d) about 5 mM to about 200 mM of arginine, proline,phenylalanine, alanine, glycine, lysine, glutamic acid, aspartic acid ormethionine; (e) about 0.01 M to about 0.1 M of hydrochloric acid (“HCl”)in an amount sufficient to achieve a pH in the range of about 5.5 toabout 7.5; and (f) a liquid carrier including but not limited to sterilewater, petroleum, animal oil, vegetable oil, mineral oil, synthetic oil,physiological saline solution, dextrose or other saccharide solution orglycols, such as ethylene glycol, propylene glycol or polyethyleneglycol; wherein said pharmaceutical composition has a pH in the range ofabout 5.5 to about 7.5; and wherein said pharmaceutical compositionoptionally comprises about 0.01% to about 1% w/v of the formulation of anon-ionic surfactant (including but not limited to Polysorbate-80 (Tween80™), Polysorbate-60 (Tween 60™), Polysorbate-40 (Tween 40™), andPolysorbate-20 (Tween 20™), polyoxyethylene alkyl ethers, including butnot limited to Brij 58™, Brij35™, as well as others such as TritonX-100™, Triton X-114™, NP40™, Span 85 and the Pluronic series ofnon-ionic surfactants (e.g., Pluronic 121)).
 28. The composition ofclaim 27 which comprises: (a) about 50 mg/mL to about 200 mg/mL of thePCSK9-specific antagonist; (b) about 1% to about 6% w/v of mannitol,trehalose or sucrose; (c) about 10 mM to about 150 mM of histidine; (d)about 10 mM to about 150 mM of arginine or proline; (e) about 0.003 M toabout 0.005 M of hydrochloric acid (“HCl”) in an amount sufficient toachieve a pH in the range of about 5.8 to about 6.5; and (f) a liquidcarrier including but not limited to sterile water; petroleum, animaloil, vegetable oil, mineral oil, synthetic oil, physiological salinesolution dextrose, or other saccharide solution or glycols, such asethylene glycol, propylene glycol or polyethylene glycol; wherein saidpharmaceutical composition has a pH in the range of about 5.8 to about6.5; and wherein said pharmaceutical composition optionally comprisesabout 0.01% to about 1% w/v of Polysorbate-80 (Tween 80™) orPolysorbate-20 (Tween 20™).
 29. The composition of claim 28 whichcomprises: (a) about 50 mg/mL to about 200 mg/mL of the PCSK9-specificantagonist; (b) about 2% to about 6% w/v of sucrose; (c) about 25 mM toabout 100 mM of histidine; (d) about 25 mM to about 100 mM of arginine;(e) about 0.0040 M to about 0.0045 M of hydrochloric acid (“HCl”) in anamount sufficient to achieve a pH in the range of about 6; and (f)sterile water; wherein said pharmaceutical composition has a pH in therange of about 6; and wherein said pharmaceutical composition optionallycomprises about 0.01% to about 1% w/v of Polysorbate-80 (Tween 80™) orPolysorbate-20 (Tween 20™).
 30. The composition of claim 29 whichcomprises: (a) about 50 mg/mL to about 200 mg/mL of the PCSK9-specificantagonist; (b) sucrose, histidine and arginine in one of the followingamounts: (i) about 3% w/v sucrose, about 50 mM histidine and about 50 mMarginine; or (ii) about 6% w/v sucrose, about 100 mM histidine and about100 mM arginine; (c) about 0.0040 M to about 0.0045 M of hydrochloricacid (“HCl”) in an amount sufficient to achieve a pH in the range ofabout 6; and (d) sterile water; wherein said pharmaceutical compositionhas a pH in the range of about 6; and wherein said pharmaceuticalcomposition optionally comprises about 0.01% to about 1% w/v ofPolysorbate-80 (Tween 80™) or Polysorbate-20 (Tween 20™).
 31. A methodfor antagonizing PCSK9 function which comprises administering aPCSK9-specific antagonist of claim
 1. 32. Use of a PCSK9-specificantagonist of claim 1 in the manufacture of a medicament forameliorating a disorder, condition or disease caused and/or exacerbatedby PCSK9 function.
 33. Isolated nucleic acid encoding a PCSK9-specificantagonist of claim
 1. 34. Isolated nucleic acid which encodes aPCSK9-specific antagonist of claim 1; wherein the CDR3 domain of theheavy chain variable region is encoded by a nucleotide sequencecomprising SEQ ID NO: 18; and/or wherein the CDR3 domain of the lightchain variable region is encoded by a nucleotide sequence comprising SEQID NO:
 8. 35. The isolated nucleic acid of claim 34 which furthercomprises: (a) CDR1 and/or CDR2 domains in the heavy chain variableregion that are encoded, respectively, by a nucleotide sequencecomprising SEQ ID NO: 14 and SEQ ID NO: 16; and/or (b) CDR1 and/or CDR2domains in the light chain variable region that are encoded,respectively, by a nucleotide sequence comprising SEQ ID NO: 4 and SEQID NO:
 6. 36. Isolated nucleic acid which encodes a PCSK9-specificantagonist of claim 1; wherein said PCSK9-specific antagonist comprises:(a) a heavy chain variable region encoded by a nucleotide sequencecomprising SEQ ID NO: 12; and/or (b) a light chain variable regionencoded by a nucleotide sequence comprising SEQ ID NO:
 28. 37. Isolatednucleic acid which encodes a PCSK9-specific antagonist of claim 1;wherein said PCSK9-specific antagonist comprises: (a) a heavy chainregion encoded by a nucleotide sequence comprising SEQ ID NO: 29; and/or(b) a light chain region encoded by a nucleotide sequence comprising SEQID NO:
 30. 38. A vector comprising nucleic acid of claim
 33. 39. Anisolated host cell or population of host cells in vitro or in situcomprising nucleic acid of claim
 33. 40. A method for producing aPCSK9-specific antagonist which comprises: (a) culturing the cell(s) ofclaim 39 under conditions appropriate for production of thePCSK9-specific antagonist; and (b) isolating the PCSK9-specificantagonist produced.
 41. An isolated host cell or population of hostcells in vitro or in situ comprising a PCSK9-specific antagonist ofclaim 1.